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Sample records for reduces endoplasmic reticulum

  1. Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces β Cell Proliferation.

    PubMed

    Szabat, Marta; Page, Melissa M; Panzhinskiy, Evgeniy; Skovsø, Søs; Mojibian, Majid; Fernandez-Tajes, Juan; Bruin, Jennifer E; Bround, Michael J; Lee, Jason T C; Xu, Eric E; Taghizadeh, Farnaz; O'Dwyer, Shannon; van de Bunt, Martijn; Moon, Kyung-Mee; Sinha, Sunita; Han, Jun; Fan, Yong; Lynn, Francis C; Trucco, Massimo; Borchers, Christoph H; Foster, Leonard J; Nislow, Corey; Kieffer, Timothy J; Johnson, James D

    2016-01-12

    Pancreatic β cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive β cells into more pliable states with reduced cellular insulin levels, increased β cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, β cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses β cell proliferation in a cell-autonomous manner.

  2. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress.

    PubMed

    Zhang, Chao; Wang, Chendan; Ren, Jianbo; Guo, Xiangjie; Yun, Keming

    2016-10-24

    Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca(2+) release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca(2+) release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca(2+) overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca(2+) release and ER stress.

  3. Glucose-6-phosphate Reduces Calcium Accumulation in Rat Brain Endoplasmic Reticulum

    DTIC Science & Technology

    2012-04-01

    low millimolar range. Most Ca2+ is sequestered within organelles , including the endoplasmic reticulum (ER), Golgi, mitochondria , and nucleus (Carafoli...G6P and thapsigargin caused generalized reduction in Ca2+ accumulation in remarkably similar patterns with no apparent gray matter regional...with glucose-6-phosphate (10 mM) or thapsigargin (1 µM), revealed very similar pattern of generalized reduction in 45Ca2+ accumulation in gray and

  4. Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress

    PubMed Central

    Zhang, Zhiguo; Li, Bo; Meng, Xiangjian; Yao, Shuangshuang; Jin, Lina; Yang, Jian; Wang, Jiqiu; Zhang, Huizhi; Zhang, Zhijian; Cai, Dongsheng; Zhang, Yifei; Ning, Guang

    2016-01-01

    The histological spectrum of nonalcoholic fatty liver diseases (NAFLD) ranges from hepatic steatosis to steatohepatitis and fibrosis. Berberine (BBR) is known for its therapeutic effect on obesity, hyperglycaemia and dyslipidaemia; however, its effect on NAFLD has yet to be thoroughly explored. Db/db mice and methionine-choline-deficient diet-fed mice were administered BBR via gavage. We found that BBR-treated mice were more resistant to steatosis in the liver than vehicle-treated mice and that BBR significantly reduced hepatic inflammation, fibrosis and lipid peroxides. The beneficial effect of BBR was associated with suppressing endoplasmic reticulum (ER) stress. Additionally, BBR decreased the free fatty acid-induced lipid accumulation and tunicamycin-induced ER stress in primary hepatocytes and hepatocyte cell lines. We demonstrated that BBR exhibited chaperone activity, reduced protein aggregation in vitro and alleviated tunicamycin-induced triglyceride and collagen deposition in vivo. Finally, we showed that BBR could reverse ER stress-activated lipogenesis through the ATF6/SREBP-1c pathway in vitro. These results indicated that BBR may be a new therapeutic strategy against hepatic steatosis and non-alcoholic steatohepatitis. PMID:26857750

  5. Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

    NASA Astrophysics Data System (ADS)

    Gunduz, Nuray; Ceylan, Hakan; Guler, Mustafa O.; Tekinay, Ayse B.

    2017-02-01

    Understanding the toxicity of nanomaterials remains largely limited to acute cellular response, i.e., short-term in vitro cell-death based assays, and analyses of tissue- and organ-level accumulation and clearance patterns in animal models, which have produced very little information about how these materials (from the toxicity point of view) interact with the complex intracellular machinery. In particular, understanding the mechanism of toxicity caused by the gradual accumulation of nanomaterials due to prolonged exposure times is essential yet still continue to be a largely unexplored territory. Herein, we show intracellular accumulation and the associated toxicity of gold nanoparticles (AuNPs) for over two-months in the cultured vascular endothelial cells. We observed that steady exposure of AuNPs at low (non-lethal) dose leads to rapid intracellular accumulation without causing any detectable cell death while resulting in elevated endoplasmic reticulum (ER) stress. Above a certain intracellular AuNP threshold, inhibition of macropinocytosis mechanism ceases further nanoparticle uptake. Interestingly, the intracellular depletion of nanoparticles is irreversible. Once reaching the maximum achievable intracellular dose, a steady depletion is observed, while no cell death is observed at any stage of this overall process. This depletion is important for reducing the ER stress. To our knowledge, this is the first report suggesting active regulation of nanoparticle uptake by cells and the impact of long-term exposure to nanoparticles in vitro.

  6. Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

    PubMed Central

    Gunduz, Nuray; Ceylan, Hakan; Guler, Mustafa O.; Tekinay, Ayse B.

    2017-01-01

    Understanding the toxicity of nanomaterials remains largely limited to acute cellular response, i.e., short-term in vitro cell-death based assays, and analyses of tissue- and organ-level accumulation and clearance patterns in animal models, which have produced very little information about how these materials (from the toxicity point of view) interact with the complex intracellular machinery. In particular, understanding the mechanism of toxicity caused by the gradual accumulation of nanomaterials due to prolonged exposure times is essential yet still continue to be a largely unexplored territory. Herein, we show intracellular accumulation and the associated toxicity of gold nanoparticles (AuNPs) for over two-months in the cultured vascular endothelial cells. We observed that steady exposure of AuNPs at low (non-lethal) dose leads to rapid intracellular accumulation without causing any detectable cell death while resulting in elevated endoplasmic reticulum (ER) stress. Above a certain intracellular AuNP threshold, inhibition of macropinocytosis mechanism ceases further nanoparticle uptake. Interestingly, the intracellular depletion of nanoparticles is irreversible. Once reaching the maximum achievable intracellular dose, a steady depletion is observed, while no cell death is observed at any stage of this overall process. This depletion is important for reducing the ER stress. To our knowledge, this is the first report suggesting active regulation of nanoparticle uptake by cells and the impact of long-term exposure to nanoparticles in vitro. PMID:28145529

  7. IGL-1 solution reduces endoplasmic reticulum stress and apoptosis in rat liver transplantation

    PubMed Central

    Mosbah, I B; Zaouali, M A; Martel, C; Bjaoui, M; Abdennebi, H B; Hotter, G; Brenner, C; Roselló-Catafau, J

    2012-01-01

    Injury due to cold ischemia reperfusion (I/R) is a major cause of primary graft non-function following liver transplantation. We postulated that I/R-induced cellular damage during liver transplantation might affect the secretory pathway, particularly at the endoplasmic reticulum (ER). We examined the involvement of ER stress in organ preservation, and compared cold storage in University of Wisconsin (UW) solution and in Institute Georges Lopez-1 (IGL-1) solution. In one group of rats, livers were preserved in UW solution for 8 h at 4 °C, and then orthotopic liver transplantation was performed according to Kamada's cuff technique. In another group, livers were preserved in IGL-1 solution. The effect of each preservation solution on the induction of ER stress, hepatic injury, mitochondrial damage and cell death was evaluated. As expected, we found increased ER stress after liver transplantation. IGL-1 solution significantly attenuated ER damage by reducing the activation of three pathways of unfolded protein response and their effector molecules caspase-12, C/EBP homologous protein-10, X-box-binding protein 1, tumor necrosis factor-associated factor 2 and eukaryotic translation initiation factor 2. This attenuation of ER stress was associated with a reduction in hepatic injury and cell death. Our results show that IGL-1 solution may be a useful means to circumvent excessive ER stress reactions associated with liver transplantation, and may optimize graft quality. PMID:22402603

  8. Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis.

    PubMed

    Seyhun, Ersin; Malo, Antje; Schäfer, Claus; Moskaluk, Christopher A; Hoffmann, Ralf-Thorsten; Göke, Burkhard; Kubisch, Constanze H

    2011-11-01

    In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.

  9. Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

    SciTech Connect

    Lee, Yea-Jin; Kim, Sung-Jo; Heo, Tae-Hwe

    2011-09-23

    Highlights: {yields} Catechin reduces the expression level of ER stress marker protein in type I Gaucher disease cells. {yields} Catechin induces the proliferation rate of GD cells similar levels to normal cells. {yields} Catechin improves wound healing activity. {yields} Catechin-mediated reductions in ER stress may be associated with enhanced cell survival. {yields} We identified catechin as a protective agent against ER stress in GD cells. -- Abstract: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset in adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.

  10. Vertical sleeve gastrectomy reduces blood pressure and hypothalamic endoplasmic reticulum stress in mice.

    PubMed

    McGavigan, Anne K; Henseler, Zachariah M; Garibay, Darline; Butler, Scott D; Jayasinghe, Sisitha; Ley, Ruth E; Davisson, Robin L; Cummings, Bethany P

    2017-03-01

    Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes remarkable improvements in cardiometabolic health, including hypertension remission. However, the mechanisms responsible remain undefined and poorly studied. Therefore, we developed and validated the first murine model of VSG that recapitulates the blood pressure-lowering effect of VSG using gold-standard radiotelemetry technology. We used this model to investigate several potential mechanisms, including body mass, brain endoplasmic reticulum (ER) stress signaling and brain inflammatory signaling, which are all critical contributors to the pathogenesis of obesity-associated hypertension. Mice fed on a high-fat diet underwent sham or VSG surgery and radiotelemeter implantation. Sham mice were fed ad libitum or were food restricted to match their body mass to VSG-operated mice to determine the role of body mass in the ability of VSG to lower blood pressure. Blood pressure was then measured in freely moving unstressed mice by radiotelemetry. VSG decreased energy intake, body mass and fat mass. Mean arterial blood pressure (MAP) was reduced in VSG-operated mice compared with both sham-operated groups. VSG-induced reductions in MAP were accompanied by a body mass-independent decrease in hypothalamic ER stress, hypothalamic inflammation and sympathetic nervous system tone. Assessment of gut microbial populations revealed VSG-induced increases in the relative abundance of Gammaproteobacteria and Enterococcus, and decreases in Adlercreutzia These results suggest that VSG reduces blood pressure, but this is only partly due to the reduction in body weight. VSG-induced reductions in blood pressure may be driven by a decrease in hypothalamic ER stress and inflammatory signaling, and shifts in gut microbial populations.

  11. Vertical sleeve gastrectomy reduces blood pressure and hypothalamic endoplasmic reticulum stress in mice

    PubMed Central

    McGavigan, Anne K.; Henseler, Zachariah M.; Garibay, Darline; Butler, Scott D.; Jayasinghe, Sisitha; Ley, Ruth E.; Davisson, Robin L.

    2017-01-01

    ABSTRACT Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes remarkable improvements in cardiometabolic health, including hypertension remission. However, the mechanisms responsible remain undefined and poorly studied. Therefore, we developed and validated the first murine model of VSG that recapitulates the blood pressure-lowering effect of VSG using gold-standard radiotelemetry technology. We used this model to investigate several potential mechanisms, including body mass, brain endoplasmic reticulum (ER) stress signaling and brain inflammatory signaling, which are all critical contributors to the pathogenesis of obesity-associated hypertension. Mice fed on a high-fat diet underwent sham or VSG surgery and radiotelemeter implantation. Sham mice were fed ad libitum or were food restricted to match their body mass to VSG-operated mice to determine the role of body mass in the ability of VSG to lower blood pressure. Blood pressure was then measured in freely moving unstressed mice by radiotelemetry. VSG decreased energy intake, body mass and fat mass. Mean arterial blood pressure (MAP) was reduced in VSG-operated mice compared with both sham-operated groups. VSG-induced reductions in MAP were accompanied by a body mass-independent decrease in hypothalamic ER stress, hypothalamic inflammation and sympathetic nervous system tone. Assessment of gut microbial populations revealed VSG-induced increases in the relative abundance of Gammaproteobacteria and Enterococcus, and decreases in Adlercreutzia. These results suggest that VSG reduces blood pressure, but this is only partly due to the reduction in body weight. VSG-induced reductions in blood pressure may be driven by a decrease in hypothalamic ER stress and inflammatory signaling, and shifts in gut microbial populations. PMID:28093508

  12. Calcium homoeostasis modulator 1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers ER stress.

    PubMed

    Gallego-Sandín, Sonia; Alonso, María Teresa; García-Sancho, Javier

    2011-08-01

    CALHM1 (calcium homoeostasis modulator 1), a membrane protein with similarity to NMDA (N-methyl-D-aspartate) receptor channels that localizes in the plasma membrane and the ER (endoplasmic reticulum) of neurons, has been shown to generate a plasma-membrane Ca(2+) conductance and has been proposed to influence Alzheimer's disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca(2+) handling in HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells by using targeted aequorins for selective monitorization of Ca(2+) transport by organelles. We find that CALHM1 increases Ca(2+) leak from the ER and, more importantly, reduces ER Ca(2+) uptake by decreasing both the transport capacity and the Ca(2+) affinity of SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase). As a result, the Ca(2+) content of the ER is drastically decreased. This reduction in the Ca(2+) content of the ER triggered the UPR (unfolded protein response) with induction of several ER stress markers, such as CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], ERdj4, GRP78 (glucose-regulated protein of 78 kDa) and XBP1 (X-box-binding protein 1). Thus CALHM1 might provide a relevant link between Ca(2+) homoeostasis disruption, ER stress and cell damage in the pathogenesis of neurodegenerative diseases.

  13. The human alpha defensin HD5 neutralizes JC polyomavirus infection by reducing endoplasmic reticulum traffic and stabilizing the viral capsid.

    PubMed

    Zins, Stephen R; Nelson, Christian D S; Maginnis, Melissa S; Banerjee, Rahul; O'Hara, Bethany A; Atwood, Walter J

    2014-01-01

    Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins.

  14. The Human Alpha Defensin HD5 Neutralizes JC Polyomavirus Infection by Reducing Endoplasmic Reticulum Traffic and Stabilizing the Viral Capsid

    PubMed Central

    Zins, Stephen R.; Nelson, Christian D. S.; Maginnis, Melissa S.; Banerjee, Rahul; O'Hara, Bethany A.

    2014-01-01

    Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins. PMID:24198413

  15. Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

    PubMed Central

    Harvey, Lloyd D.; Yin, Yan; Attarwala, Insiya Y.; Begum, Gulnaz; Deng, Julia; Yan, Hong Q.; Dixon, C. Edward

    2015-01-01

    We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1+ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32+ microglia or macrophages, but an increased CD206+ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1+ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1+ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to

  16. Endoplasmic reticulum aminopeptidases: biochemistry, physiology and pathology.

    PubMed

    Hattori, Akira; Tsujimoto, Masafumi

    2013-09-01

    The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized.

  17. Increased classical endoplasmic reticulum stress is sufficient to reduce chondrocyte proliferation rate in the growth plate and decrease bone growth.

    PubMed

    Kung, Louise H W; Rajpar, M Helen; Preziosi, Richard; Briggs, Michael D; Boot-Handford, Raymond P

    2015-01-01

    Mutations in genes encoding cartilage oligomeric matrix protein and matrilin-3 cause a spectrum of chondrodysplasias called multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). The majority of these diseases feature classical endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) as a result of misfolding of the mutant protein. However, the importance and the pathological contribution of ER stress in the disease pathogenesis are unknown. The aim of this study was to investigate the generic role of ER stress and the UPR in the pathogenesis of these diseases. A transgenic mouse line (ColIITgcog) was generated using the collagen II promoter to drive expression of an ER stress-inducing protein (Tgcog) in chondrocytes. The skeletal and histological phenotypes of these ColIITgcog mice were characterised. The expression and intracellular retention of Tgcog induced ER stress and activated the UPR as characterised by increased BiP expression, phosphorylation of eIF2α and spliced Xbp1. ColIITgcog mice exhibited decreased long bone growth and decreased chondrocyte proliferation rate. However, there was no disruption of chondrocyte morphology or growth plate architecture and perturbations in apoptosis were not apparent. Our data demonstrate that the targeted induction of ER stress in chondrocytes was sufficient to reduce the rate of bone growth, a key clinical feature associated with MED and PSACH, in the absence of any growth plate dysplasia. This study establishes that classical ER stress is a pathogenic factor that contributes to the disease mechanism of MED and PSACH. However, not all the pathological features of MED and PSACH were recapitulated, suggesting that a combination of intra- and extra-cellular factors are likely to be responsible for the disease pathology as a whole.

  18. Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.

    PubMed

    Montague, Karli; Malik, Bilal; Gray, Anna L; La Spada, Albert R; Hanna, Michael G; Szabadkai, Gyorgy; Greensmith, Linda

    2014-07-01

    Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.

  19. Endoplasmic Reticulum Stress Regulates Adipocyte Resistin Expression

    PubMed Central

    Lefterova, Martina I.; Mullican, Shannon E.; Tomaru, Takuya; Qatanani, Mohammed; Schupp, Michael; Lazar, Mitchell A.

    2009-01-01

    OBJECTIVE Resistin is a secreted polypeptide that impairs glucose metabolism and, in rodents, is derived exclusively from adipocytes. In murine obesity, resistin circulates at elevated levels but its gene expression in adipose tissue is paradoxically reduced. The mechanism behind the downregulation of resistin mRNA is poorly understood. We investigated whether endoplasmic reticulum (ER) stress, which is characteristic of obese adipose tissue, regulates resistin expression in cultured mouse adipocytes. RESEARCH DESIGN AND METHODS The effects of endoplasmic stress inducers on resistin mRNA and secreted protein levels were examined in differentiated 3T3-L1 adipocytes, focusing on the expression and genomic binding of transcriptional regulators of resistin. The association between downregulated resistin mRNA and induction of ER stress was also investigated in the adipose tissue of mice fed a high-fat diet. RESULTS ER stress reduced resistin mRNA in 3T3-L1 adipocytes in a time- and dose-dependent manner. The effects of ER stress were transcriptional because of downregulation of CAAT/enhancer binding protein-α and peroxisome proliferator–activated receptor-γ transcriptional activators and upregulation of the transcriptional repressor CAAT/enhancer binding protein homologous protein-10 (CHOP10). Resistin protein was also substantially downregulated, showing a close correspondence with mRNA levels in 3T3-L1 adipocytes as well as in the fat pads of obese mice. CONCLUSIONS ER stress is a potent regulator of resistin, suggesting that ER stress may underlie the local downregulation of resistin mRNA and protein in fat in murine obesity. The paradoxical increase in plasma may be because of various systemic abnormalities associated with obesity and insulin resistance. PMID:19491212

  20. Endoplasmic Reticulum Stress and Associated ROS

    PubMed Central

    Zeeshan, Hafiz Maher Ali; Lee, Geum Hwa; Kim, Hyung-Ryong; Chae, Han-Jung

    2016-01-01

    The endoplasmic reticulum (ER) is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS). Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI)-endoplasmic reticulum oxidoreductin (ERO)-1, glutathione (GSH)/glutathione disuphide (GSSG), NADPH oxidase 4 (Nox4), NADPH-P450 reductase (NPR), and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases. PMID:26950115

  1. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  2. Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells.

    PubMed

    Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin; Jeong, Jae-Hoon; Kang, Seongman; Lim, Young-Bin

    2014-07-25

    Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.

  3. 3-O-Hydroxytyrosol glucuronide and 4-O-hydroxytyrosol glucuronide reduce endoplasmic reticulum stress in vitro.

    PubMed

    Giordano, Elena; Dangles, Olivier; Rakotomanomana, Njara; Baracchini, Silvia; Visioli, Francesco

    2015-10-01

    Endoplasmic reticulum (ER) stress is important for atherosclerosis development and is mediated by the unfolded protein response (UPR). In this work, we synthesized two among the most physiologically-prominent hydroxytyrosol HT hepatic metabolites, i.e. 3-O-HT glucuronide and 4-O-HT glucuronide and we tested their activities on ER stress (in human hepatocarcinoma HepG2 cells), to gain further insight into the cardiopreventive properties of HT, extra virgin olive oil, and the Mediterranean diet. We report that 3-O-HT glucuronide and 4-O-HT glucuronide inhibit tunicamycin-induced ER stress. As compared with the effects of the parent molecule, 3-O-HT glucuronide and 4-O-HT glucuronide at 10 μM and 25 μM alone induced a milder change in mRNA expression levels of both CCAAT-enhancer-binding protein homologous protein (CHOP) and glucose regulated protein GRP78 immunoglobulin heavy chain binding protein (BiP). In conclusion, we add further evidence to the hypothesis that the HT intake might be atheroprotective and reiterate the usefulness to preferably use high-quality, high-(poly)phenol extra virgin olive oil as a prominent condiment.

  4. Dietary Cocoa Powder Improves Hyperlipidemia and Reduces Atherosclerosis in apoE Deficient Mice through the Inhibition of Hepatic Endoplasmic Reticulum Stress

    PubMed Central

    Guan, Hua; Lin, Yan; Bai, Liang; An, Yingfeng; Shang, Jianan; Wang, Zhao; Zhao, Sihai; Fan, Jianglin

    2016-01-01

    Cocoa powder is rich in flavonoids, which have many beneficial effects on human health, including antioxidative and anti-inflammatory effects. The aim of our study was to investigate whether the intake of cocoa powder has any influence on hyperlipidemia and atherosclerosis and examine the underlying molecular mechanisms. We fed apoE knockout mice a Western diet supplemented with either 0.2% (low group) or 2% (high group) cocoa powder for 12 weeks. The groups fed dietary cocoa powder showed a significant reduction in both plasma cholesterol levels and aortic atherosclerosis compared to the control group. Analysis of mRNA profiling of aortic atherosclerotic lesions revealed that the expression of several genes related to apoptosis, lipid metabolism, and inflammation was significantly reduced, while the antiapoptotic gene Bcl2 was significantly increased in the cocoa powder group compared to the control. RT-PCR analysis along with Western blotting revealed that a diet containing cocoa powder inhibited the expression of hepatic endoplasmic reticulum stress. These data suggest that cocoa powder intake improves hyperlipidemia and atherosclerosis, and such beneficial effects are possibly mediated through the suppression of hepatic endoplasmic reticulum stress. PMID:26980943

  5. Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

    SciTech Connect

    Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin; Jeong, Jae-Hoon; Kang, Seongman; Lim, Young-Bin

    2014-07-25

    Highlights: • UPR activation precedes caspase activation in irradiated IEC-6 cells. • Chemical ER stress inducers radiosensitize IEC-6 cells. • siRNAs that targeted ER stress responses ameliorate IR-induced cell death. • Chemical chaperons prevent cell death in irradiated IEC-6 cells. - Abstract: Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.

  6. Obesity and endoplasmic reticulum (ER) stresses

    PubMed Central

    Tripathi, Yamini B.; Pandey, Vivek

    2012-01-01

    In obesity, the adipose cells behave as inflammatory source and result to low grade inflammation. This systemic inflammation along with oxidative stress is a silent killer and damages other vital organs also. High metabolic process, induced due to high nutritional intake, results to endoplasmic reticulum (ER) stress and mitochondrial stress. This review describes the triggering factor and basic mechanism behind the obesity mediated these stresses in relation to inflammation. Efforts have been made to describe the effect-response cycle between adipocytes and non-adipocyte cells with reference to metabolic syndrome (MS). PMID:22891067

  7. Protein Translocation across the Rough Endoplasmic Reticulum

    PubMed Central

    Mandon, Elisabet C.; Trueman, Steven F.; Gilmore, Reid

    2013-01-01

    The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration. PMID:23251026

  8. The endoplasmic reticulum stress response: A link with tuberculosis?

    PubMed

    Cui, Yongyong; Zhao, Deming; Barrow, Paul Andrew; Zhou, Xiangmei

    2016-03-01

    Tuberculosis (TB) remains a major cause of mortality and morbidity in the worldwide. The endoplasmic-reticulum stress (ERS) response constitutes a cellular process that is triggered by mycobacterial infection that disturbs the folding of proteins in the endoplasmic reticulum (ER). The unfolded protein response (UPR) is induced to suspend the synthesis of early proteins and reduce the accumulation of unfolded- or misfolded proteins in the ER restoring normal physiological cell function. Prolonged or uncontrolled ERS leads to the activation of three signaling pathways (IRE1, PERK and ATF6) which directs the cell towards apoptosis. The absence of this process facilitates spread of the mycobacteria within the body. We summarize here recent advances in understanding the signaling pathway diversity governing ERS in relation to TB.

  9. An endoplasmic reticulum-specific cyclophilin.

    PubMed Central

    Hasel, K W; Glass, J R; Godbout, M; Sutcliffe, J G

    1991-01-01

    Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression. Images PMID:1710767

  10. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

    PubMed Central

    Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245

  11. Endoplasmic-Reticulum Calcium Depletion and Disease

    PubMed Central

    Mekahli, Djalila; Bultynck, Geert; Parys, Jan B.; De Smedt, Humbert; Missiaen, Ludwig

    2011-01-01

    The endoplasmic reticulum (ER) as an intracellular Ca2+ store not only sets up cytosolic Ca2+ signals, but, among other functions, also assembles and folds newly synthesized proteins. Alterations in ER homeostasis, including severe Ca2+ depletion, are an upstream event in the pathophysiology of many diseases. On the one hand, insufficient release of activator Ca2+ may no longer sustain essential cell functions. On the other hand, loss of luminal Ca2+ causes ER stress and activates an unfolded protein response, which, depending on the duration and severity of the stress, can reestablish normal ER function or lead to cell death. We will review these various diseases by mainly focusing on the mechanisms that cause ER Ca2+ depletion. PMID:21441595

  12. Endoplasmic Reticulum Stress Response in Arabidopsis Roots

    PubMed Central

    Cho, Yueh; Kanehara, Kazue

    2017-01-01

    Roots are the frontier of plant body to perceive underground environmental change. Endoplasmic reticulum (ER) stress response represents circumvention of cellular stress caused by various environmental changes; however, a limited number of studies are available on the ER stress responses in roots. Here, we report the tunicamycin (TM) -induced ER stress response in Arabidopsis roots by monitoring expression patterns of immunoglobulin-binding protein 3 (BiP3), a representative marker for the response. Roots promptly responded to the TM-induced ER stress through the induction of similar sets of ER stress-responsive genes. However, not all cells responded uniformly to the TM-induced ER stress in roots, as BiP3 was highly expressed in root tips, an outer layer in elongation zone, and an inner layer in mature zone of roots. We suggest that ER stress response in roots has tissue specificity. PMID:28298914

  13. Structural organization of the endoplasmic reticulum

    PubMed Central

    Voeltz, Gia K.; Rolls, Melissa M.; Rapoport, Tom A.

    2002-01-01

    The endoplasmic reticulum (ER) is a continuous membrane system but consists of various domains that perform different functions. Structurally distinct domains of this organelle include the nuclear envelope (NE), the rough and smooth ER, and the regions that contact other organelles. The establishment of these domains and the targeting of proteins to them are understood to varying degrees. Despite its complexity, the ER is a dynamic structure. In mitosis it must be divided between daughter cells and domains must be re-established, and even in interphase it is constantly rearranged as tubules extend along the cytoskeleton. Throughout these rearrangements the ER maintains its basic structure. How this is accomplished remains mysterious, but some insight has been gained from in vitro systems. PMID:12370207

  14. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

    PubMed Central

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-01-01

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

  15. Endoplasmic Reticulum Stress and Ethanol Neurotoxicity.

    PubMed

    Yang, Fanmuyi; Luo, Jia

    2015-10-14

    Ethanol abuse affects virtually all organ systems and the central nervous system (CNS) is particularly vulnerable to excessive ethanol exposure. Ethanol exposure causes profound damages to both the adult and developing brain. Prenatal ethanol exposure induces fetal alcohol spectrum disorders (FASD) which is associated with mental retardation and other behavioral deficits. A number of potential mechanisms have been proposed for ethanol-induced brain damage; these include the promotion of neuroinflammation, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, and thiamine deficiency. The endoplasmic reticulum (ER) regulates posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and induces unfolded protein response (UPR) which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). UPR is initiated to protect cells from overwhelming ER protein loading. However, sustained ER stress may result in cell death. ER stress has been implied in various CNS injuries, including brain ischemia, traumatic brain injury, and aging-associated neurodegeneration, such as Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). However, effects of ethanol on ER stress in the CNS receive less attention. In this review, we discuss recent progress in the study of ER stress in ethanol-induced neurotoxicity. We also examine the potential mechanisms underlying ethanol-mediated ER stress and the interaction among ER stress, oxidative stress and autophagy in the context of ethanol neurotoxicity.

  16. Endoplasmic reticulum stress regulates rat mandibular cartilage thinning under compressive mechanical stress.

    PubMed

    Li, Huang; Zhang, Xiang-Yu; Wu, Tuo-Jiang; Cheng, Wei; Liu, Xin; Jiang, Ting-Ting; Wen, Juan; Li, Jie; Ma, Qiao-Ling; Hua, Zi-Chun

    2013-06-21

    Compressive mechanical stress-induced cartilage thinning has been characterized as a key step in the progression of temporomandibular joint diseases, such as osteoarthritis. However, the regulatory mechanisms underlying this loss have not been thoroughly studied. Here, we used an established animal model for loading compressive mechanical stress to induce cartilage thinning in vivo. The mechanically stressed mandibular chondrocytes were then isolated to screen potential candidates using a proteomics approach. A total of 28 proteins were identified that were directly or indirectly associated with endoplasmic reticulum stress, including protein disulfide-isomerase, calreticulin, translationally controlled tumor protein, and peptidyl-prolyl cis/trans-isomerase protein. The altered expression of these candidates was validated at both the mRNA and protein levels. The induction of endoplasmic reticulum stress by mechanical stress loading was confirmed by the activation of endoplasmic reticulum stress markers, the elevation of the cytoplasmic Ca(2+) level, and the expansion of endoplasmic reticulum membranes. More importantly, the use of a selective inhibitor to block endoplasmic reticulum stress in vivo reduced the apoptosis observed at the early stages of mechanical stress loading and inhibited the proliferation observed at the later stages of mechanical stress loading. Accordingly, the use of the inhibitor significantly restored cartilage thinning. Taken together, these results demonstrated that endoplasmic reticulum stress is significantly activated in mechanical stress-induced mandibular cartilage thinning and, more importantly, that endoplasmic reticulum stress inhibition alleviates this loss, suggesting a novel pharmaceutical strategy for the treatment of mechanical stress-induced temporomandibular joint diseases.

  17. Development of Endoplasmic Reticulum Stress during Experimental Oxalate Nephrolithiasis.

    PubMed

    Motin, Yu G; Lepilov, A V; Bgatova, N P; Zharikov, A Yu; Motina, N V; Lapii, G A; Lushnikova, E L; Nepomnyashchikh, L M

    2016-01-01

    Morphological and ultrastructural study of the kidney was performed in rats with oxalate nephrolithiasis. Specific features of endoplasmic reticulum stress were evaluated during nephrolithiasis and treatment with α-tocopherol. We observed the signs of endoplasmic reticulum stress with activation of proapoptotic pathways and injury to the cell lining in nephron tubules and collecting ducts. Ultrastructural changes were found in the organelles, nuclei, and cell membranes of epitheliocytes. A relationship was revealed between endoplasmic reticulum stress and oxidative damage, which developed at the early state of lithogenesis.

  18. Endoplasmic reticulum stress causes EBV lytic replication

    PubMed Central

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K.; Rowe, David T.; Wadowsky, Robert M.

    2011-01-01

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)–specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress–dependent and –independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)–treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress. PMID:21849482

  19. Endoplasmic Reticulum Stress Plays a Key Role in the Pathogenesis of Diabetic Peripheral Neuropathy

    PubMed Central

    Lupachyk, Sergey; Watcho, Pierre; Stavniichuk, Roman; Shevalye, Hanna; Obrosova, Irina G.

    2013-01-01

    Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins impairs metabolism, transcriptional regulation, and gene expression, and it is a key mechanism of cell injury. Endoplasmic reticulum stress plays an important role in cardiovascular and neurodegenerative diseases, cancer, and diabetes. We evaluated the role for this phenomenon in diabetic peripheral neuropathy. Endoplasmic reticulum stress manifest in upregulation of multiple components of unfolded protein response was identified in neural tissues (sciatic nerve, spinal cord) of streptozotocin diabetic rats and mice. A chemical chaperone, trimethylamine oxide, administered for 12 weeks after induction of diabetes (110 mg⋅kg−1⋅d−1, a prevention paradigm) attenuated endoplasmic reticulum stress, peripheral nerve dysfunction, intraepidermal nerve fiber loss, and sciatic nerve and spinal cord oxidative-nitrative stress in streptozotocin diabetic rats. Similar effects on diabetes-induced endoplasmic reticulum stress and peripheral nerve dysfunction were observed with a structurally unrelated chemical chaperone, 4-phenylbutyric acid (100 mg⋅kg−1⋅d−1, intraperitoneal). CCAAT/enhancer-binding protein homologous protein (CHOP)−/− mice made diabetic with streptozotocin displayed less severe sciatic nerve oxidative-nitrative stress and peripheral neuropathy than the wild-type (C57Bl6/J) mice. Neither chemical chaperones nor CHOP gene deficiency reduced diabetic hyperglycemia. Our findings reveal an important role of endoplasmic reticulum stress in the development of diabetic peripheral neuropathy and identify a potential new therapeutic target. PMID:23364451

  20. Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin.

    PubMed

    Tufi, R; Panaretakis, T; Bianchi, K; Criollo, A; Fazi, B; Di Sano, F; Tesniere, A; Kepp, O; Paterlini-Brechot, P; Zitvogel, L; Piacentini, M; Szabadkai, G; Kroemer, G

    2008-02-01

    Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

  1. Inhibition of Brain Mitogen-Activated Protein Kinase Signaling Reduces Central Endoplasmic Reticulum Stress and Inflammation and Sympathetic Nerve Activity in Heart Failure Rats.

    PubMed

    Wei, Shun-Guang; Yu, Yang; Weiss, Robert M; Felder, Robert B

    2016-01-01

    Mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress in the brain have been implicated in the pathophysiology of hypertension. This study determined whether ER stress occurs in subfornical organ and hypothalamic paraventricular nucleus in heart failure (HF) and how MAPK signaling interacts with ER stress and other inflammatory mediators. HF rats had significantly higher levels of the ER stress biomarkers (glucose-regulated protein 78, activating transcription factor 6, activating transcription factor 4, X-box binding protein 1, P58(IPK), and C/EBP homologous protein) in subfornical organ and paraventricular nucleus, which were attenuated by a 4-week intracerebroventricular infusion of inhibitors selective for p44/42 MAPK (PD98059), p38 MAPK (SB203580), or c-Jun N-terminal kinase (SP600125). HF rats also had higher mRNA levels of tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and nuclear factor-κB p65, and a lower mRNA level of IκB-α, in subfornical organ and paraventricular nucleus, compared with SHAM rats, and these indicators of increased inflammation were attenuated in the HF rats treated with the MAPK inhibitors. Plasma norepinephrine level was higher in HF rats than in SHAM rats but was reduced in the HF rats treated with PD98059 and SB203580. A 4-week intracerebroventricular infusion of PD98059 also improved some hemodynamic and anatomic indicators of left ventricular function in HF rats. These data demonstrate that ER stress increases in the subfornical organ and paraventricular nucleus of rats with ischemia-induced HF and that inhibition of brain MAPK signaling reduces brain ER stress and inflammation and decreases sympathetic excitation in HF. An interaction between MAPK signaling and ER stress in cardiovascular regions of the brain may contribute to the development of HF.

  2. Inhibition of brain mitogen-activated protein kinase signaling reduces central endoplasmic reticulum stress and inflammation and sympathetic nerve activity in heart failure rats

    PubMed Central

    Wei, Shun-Guang; Yu, Yang; Weiss, Robert M.; Felder, Robert B.

    2015-01-01

    Mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress in the brain have been implicated in the pathophysiological mechanisms in hypertension. The present study determined whether ER stress occurs in subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN) in heart failure (HF), and how MAPK signaling interacts with ER stress and other inflammatory mediators. HF rats had significantly higher levels of the ER stress biomarkers (GRP78, ATF6, ATF4, XBP-1, P58IPK and CHOP) in SFO and PVN, which were attenuated by a 4-week intracerebroventricular (ICV) infusion of inhibitors selective for p44/42 MAPK (PD98059), p38 MAPK (SB203580) or JNK (SP600125). HF rats also had higher mRNA levels of tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2 and NF-κB p65 and lower mRNA level of IκB-α in SFO and PVN, compared with SHAM rats, and these indicators of increased inflammation were attenuated in the HF rats treated with the MAPK inhibitors. Plasma norepinephrine level was higher in HF than SHAM rats, but was reduced in the HF rats treated with PD98059 and SB203580. A 4-week ICV infusion of PD98059 also improved some hemodynamic and anatomic indicators of left ventricular function in HF rats. These data demonstrate that ER stress increases in the SFO and PVN of rats with ischemia-induced HF, and that inhibition of brain MAPK signaling reduces brain ER stress and inflammation and decreases sympathetic excitation in HF. An interaction between MAPK signaling and ER stress in cardiovascular regions of the brain may contribute to the development of HF. PMID:26573710

  3. Endoplasmic Reticulum Protein Quality Control Failure in Myelin Disorders

    PubMed Central

    Volpi, Vera G.; Touvier, Thierry; D'Antonio, Maurizio

    2017-01-01

    Reaching the correct three-dimensional structure is crucial for the proper function of a protein. The endoplasmic reticulum (ER) is the organelle where secreted and transmembrane proteins are synthesized and folded. To guarantee high fidelity of protein synthesis and maturation in the ER, cells have evolved ER-protein quality control (ERQC) systems, which assist protein folding and promptly degrade aberrant gene products. Only correctly folded proteins that pass ERQC checkpoints are allowed to exit the ER and reach their final destination. Misfolded glycoproteins are detected and targeted for degradation by the proteasome in a process known as endoplasmic reticulum-associated degradation (ERAD). The excess of unstructured proteins in the ER triggers an adaptive signal transduction pathway, called unfolded protein response (UPR), which in turn potentiates ERQC activities in order to reduce the levels of aberrant molecules. When the situation cannot be restored, the UPR drives cells to apoptosis. Myelin-forming cells of the central and peripheral nervous system (oligodendrocytes and Schwann cells) synthesize a large amount of myelin proteins and lipids and therefore are particularly susceptible to ERQC failure. Indeed, deficits in ERQC and activation of ER stress/UPR have been implicated in several myelin disorders, such as Pelizaeus-Merzbacher and Krabbe leucodystrophies, vanishing white matter disease and Charcot-Marie-Tooth neuropathies. Here we discuss recent evidence underlying the importance of proper ERQC functions in genetic disorders of myelinating glia. PMID:28101003

  4. From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration.

    PubMed

    Hoffmann, Christiane; Plocharski, Bartolome; Haferkamp, Ilka; Leroch, Michaela; Ewald, Ralph; Bauwe, Hermann; Riemer, Jan; Herrmann, Johannes M; Neuhaus, H Ekkehard

    2013-07-01

    The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO(2) concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.

  5. Altered Endoplasmic Reticulum Calcium Pump Expression during Breast Tumorigenesis

    PubMed Central

    Papp, Béla; Brouland, Jean-Philippe

    2011-01-01

    Endoplasmic reticulum calcium homeostasis is involved in several essential cell functions including cell proliferation, protein synthesis, stress responses or secretion. Calcium uptake into the endoplasmic reticulum is performed by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). In order to study endoplasmic reticulum calcium homeostasis in situ in mammary tissue, in this work SERCA3 expression was investigated in normal breast and in its benign and malignant lesions in function of the cell type, degree of malignancy, and histological and molecular parameters of the tumors. Our data indicate, that although normal breast acinar epithelial cells express SERCA3 abundantly, its expression is strongly decreased already in very early non-malignant epithelial lesions such as adenosis, and remains low in lobular carcinomas. Whereas normal duct epithelium expresses significant amounts of SERCA3, its expression is decreased in several benign ductal lesions, as well as in ductal adenocarcinoma. The loss of SERCA3 expression is correlated with Elston-Ellis grade, negative hormone receptor expression or triple negative status in ductal carcinomas. The concordance between decreased SERCA3 expression and several histological, as well as molecular markers of ductal carcinogenesis indicates that endoplasmic reticulum calcium homeostasis is remodeled during tumorigenesis in the breast epithelium. PMID:21863130

  6. Endoplasmic reticulum stress: The cause and solution to Huntington's disease?

    PubMed

    Jiang, Yuwei; Chadwick, Sarah R; Lajoie, Patrick

    2016-10-01

    Accumulation of misfolded proteins is a hallmark of many human diseases, including several incurable neurological disorders, such as Huntington's disease (HD). In HD, expansion of a polyglutamine stretch within the first exon of the Huntingtin protein (Htt) leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Several studies in various organisms (from yeast to humans) have identified the accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER stress) as a crucial determinant of cellular toxicity in HD. In this review, we highlight the recent research linking HD to ER stress. We also discuss how the modulation of signaling pathways responsible for coping with misfolded protein accumulation in the ER may constitute attractive methods to reduce toxicity and identify new therapeutic targets for treatment of HD. This article is part of a Special Issue entitled SI:ER stress.

  7. Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition.

    PubMed

    Cerqua, Cristina; Anesti, Vassiliki; Pyakurel, Aswin; Liu, Dan; Naon, Deborah; Wiche, Gerhard; Baffa, Raffaele; Dimmer, Kai S; Scorrano, Luca

    2010-11-01

    Trichoplein/mitostatin (TpMs) is a keratin-binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)-dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca(2+)-dependent stimuli that require ER-mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria-ER juxtaposition.

  8. Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.

    PubMed Central

    Terasaki, M; Slater, N T; Fein, A; Schmidek, A; Reese, T S

    1994-01-01

    Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions. Images PMID:7519781

  9. Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

    PubMed

    Chanat, Eric; Le Parc, Annabelle; Lahouassa, Hichem; Badaoui, Bouabid

    2016-06-01

    In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

  10. Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum.

    PubMed

    Zito, Ester; Buono, Mario; Pepe, Stefano; Settembre, Carmine; Annunziata, Ida; Surace, Enrico Maria; Dierks, Thomas; Monti, Maria; Cozzolino, Marianna; Pucci, Piero; Ballabio, Andrea; Cosma, Maria Pia

    2007-05-16

    Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.

  11. Endoplasmic reticulum stress: implications for inflammatory bowel disease pathogenesis

    PubMed Central

    Kaser, Arthur; Martínez-Naves, Eduardo; Blumberg, Richard S.

    2015-01-01

    Purpose of review To provide an overview of the emerging role of cellular stress responses in inflammatory bowel disease (IBD). Recent findings The unfolded protein response (UPR) is a primitive cellular pathway that is engaged when responding to endoplasmic reticulum stress and regulates autophagy. Highly secretory cells such as Paneth cells and goblet cells in the intestines are particularly susceptible to endoplasmic reticulum stress and are exceedingly dependent upon a properly functioning UPR to maintain cellular viability and homeostasis. Primary genetic abnormalities within the components of the UPR (e.g. XBP1, ARG2, ORMDL3), genes that encode proteins reliant upon a robust secretory pathway (e.g. MUC2, HLAB27) and environmental factors that create disturbances in the UPR (e.g. microbial products and inflammatory cytokines) are important factors in the primary development and/or perpetuation of intestinal inflammation. Summary Endoplasmic reticulum stress is an important new pathway involved in the development of intestinal inflammation associated with IBD and likely other intestinal inflammatory disorders. PMID:20495455

  12. Naltrexone attenuates endoplasmic reticulum stress induced hepatic injury in mice.

    PubMed

    Moslehi, A; Nabavizadeh, F; Nabavizadeh, Fatemeh; Dehpour, A R; Dehpou, A R; Tavanga, S M; Hassanzadeh, G; Zekri, A; Nahrevanian, H; Sohanaki, H

    2014-09-01

    Endoplasmic reticulum (ER) stress provides abnormalities in insulin action, inflammatory responses, lipoprotein B100 degradation and hepatic lipogenesis. Excess accumulation of triglyceride in hepatocytes may also lead to disorders such as non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Opioid peptides are involved in triglyceride and cholesterol dysregulation, inflammation and cell death. In this study, we evaluated Naltrexone effects on ER stress induced liver injury. To do so, C57/BL6 mice received saline, DMSO and Naltrexone, as control groups. ER stress was induced by tunicamycin (TM) injection. Naltrexone was given before TM administration. Liver blood flow and biochemical serum analysis were measured. Histopathological evaluations, TNF-α measurement and Real-time RT-PCR were also performed. TM challenge provokes steatosis, cellular ballooning and lobular inflammation which significantly reduced in Naltrexone treated animals. ALT, AST and TNF-α increased in the TM group and improved in the Naltrexone plus TM group. Triglyceride and cholesterol levels decreased in TM treated mice with no increase in Naltrexone treated animals. In the Naltrexone plus TM group, gene expression of Bax/Bcl-2 ratio and caspase3 significantly lowered compared with the TM group. In this study, we found that Naltrexone had a notable alleviating role in ER stress induced steatosis and liver injury.

  13. Mitochondria-endoplasmic reticulum choreography: structure and signaling dynamics.

    PubMed

    Pizzo, Paola; Pozzan, Tullio

    2007-10-01

    Mitochondria and endoplasmic reticulum (ER) have different roles in living cells but they interact both physically and functionally. A key aspect of the mitochondria-ER relationship is the modulation of Ca(2+) signaling during cell activation, which thus affects a variety of physiological processes. We focus here on the molecular aspects that control the dynamics of the organelle-organelle interaction and their relationship with Ca(2+) signals, also discussing the consequences that these phenomena have, not only for cell physiology but also in the control of cell death.

  14. Mitofusin 2 ablation increases endoplasmic reticulum-mitochondria coupling.

    PubMed

    Filadi, Riccardo; Greotti, Elisa; Turacchio, Gabriele; Luini, Alberto; Pozzan, Tullio; Pizzo, Paola

    2015-04-28

    The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER-mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca(2+) transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca(2+) overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER-mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER-mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.

  15. Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.

    PubMed

    Manfredi, Giovanni; Kawamata, Hibiki

    2016-06-01

    Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS.

  16. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori

    PubMed Central

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-01-01

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis. PMID:26556347

  17. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    SciTech Connect

    Liu Qiong; Zhan Jinbiao . E-mail: jzhan2k@zju.edu.cn; Chen Xinhong; Zheng Shu

    2006-05-12

    Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.

  18. Sigma-1 receptors (sigma(1) binding sites) form raft-like microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export.

    PubMed

    Hayashi, Teruo; Su, Tsung-Ping

    2003-08-01

    The brain sigma-1 receptors can bind neurosteroids and psychotropic drugs, including neuroleptics and cocaine and are implicated in schizophrenia, depression, and drug dependence. In this study, we found that sigma-1 receptors specifically target lipid storage sites (lipid droplets) on the endoplasmic reticulum by forming a distinct class of lipid microdomains. Both endogenously expressing sigma-1 receptors and transfected C-terminally enhanced yellow fluorescent protein (EYFP)-tagged sigma-1 receptors (Sig-1R-EYFP) target unique "ring-like" structures associated with endoplasmic reticulum reticular networks in NG108-15 cells. The ring-like structures contain neutral lipids and are enlarged by the oleate treatment, indicating that they are endoplasmic reticulum-associated lipid droplets (ER-LDs). sigma-1 receptors colocalize with caveolin-2, a cholesterol-binding protein in lipid rafts on the ER-LDs, but not with adipocyte differentiation-related protein (ADRP), a cytosolic lipid droplet (c-LD)-specific protein. When the double-arginine ER retention signal on the N terminus of sigma-1 receptors is truncated, sigma-1 receptors no longer exist on ER-LDs, but predominantly target c-LDs, which contain ADRP. sigma-1 receptors on ER-LDs form detergent-resistant raft-like lipid microdomains, the buoyancy of which is different from that of plasma membrane lipid rafts. (+)-Pentazocine causes sigma-1 receptors to disappear from the microdomains. N-Terminally EYFP-tagged sigma-1 receptors (EYFP-Sig-1R) failed to target ER-LDs. EYFP-Sig-1R-transfected cells showed an unrestricted distribution of neutral lipids all over the endoplasmic reticulum network, decreases in c-LDs and cholesterol in plasma membranes, and the bulbous aggregation of endoplasmic reticulum. Thus, sigma-1 receptors are unique endoplasmic reticulum proteins that regulate the compartmentalization of lipids on the endoplasmic reticulum and their export from the endoplasmic reticulum to plasma membrane and c-LDs.

  19. α-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions.

    PubMed

    Calì, Tito; Ottolini, Denis; Negro, Alessandro; Brini, Marisa

    2012-05-25

    α-Synuclein has a central role in Parkinson disease, but its physiological function and the mechanism leading to neuronal degeneration remain unknown. Because recent studies have highlighted a role for α-synuclein in regulating mitochondrial morphology and autophagic clearance, we investigated the effect of α-synuclein in HeLa cells on mitochondrial signaling properties focusing on Ca(2+) homeostasis, which controls essential bioenergetic functions. By using organelle-targeted Ca(2+)-sensitive aequorin probes, we demonstrated that α-synuclein positively affects Ca(2+) transfer from the endoplasmic reticulum to the mitochondria, augmenting the mitochondrial Ca(2+) transients elicited by agonists that induce endoplasmic reticulum Ca(2+) release. This effect is not dependent on the intrinsic Ca(2+) uptake capacity of mitochondria, as measured in permeabilized cells, but correlates with an increase in the number of endoplasmic reticulum-mitochondria interactions. This action specifically requires the presence of the C-terminal α-synuclein domain. Conversely, α-synuclein siRNA silencing markedly reduces mitochondrial Ca(2+) uptake, causing profound alterations in organelle morphology. The enhanced accumulation of α-synuclein into the cells causes the redistribution of α-synuclein to localized foci and, similarly to the silencing of α-synuclein, reduces the ability of mitochondria to accumulate Ca(2+). The absence of efficient Ca(2+) transfer from endoplasmic reticulum to mitochondria results in augmented autophagy that, in the long range, could compromise cellular bioenergetics. Overall, these findings demonstrate a key role for α-synuclein in the regulation of mitochondrial homeostasis in physiological conditions. Elevated α-synuclein expression and/or eventually alteration of the aggregation properties cause the redistribution of the protein within the cell and the loss of modulation on mitochondrial function.

  20. Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis

    PubMed Central

    Bravo-Sagua, R.; Rodriguez, A.E.; Kuzmicic, J.; Gutierrez, T.; Lopez-Crisosto, C.; Quiroga, C.; Díaz-Elizondo, J.; Chiong, M.; Gillette, T.G.; Rothermel, B.A.; Lavandero, S.

    2014-01-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132

  1. Characterization of Ca2+ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

    PubMed Central

    Buckhout, Thomas J.

    1984-01-01

    The characteristics of Ca2+ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca2+ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0. The Ca2+-dependent modulation protein, calmodulin, was tested for its effect on Ca2+ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca2+ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca2+ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca2+ transport activity. The inhibition of Ca2+ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca2+ uptake into root endoplasmic reticulum. PMID:16663981

  2. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

    PubMed

    Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

    2016-04-01

    It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

  3. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs

    PubMed Central

    Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W.; Schalek, Richard; Hayworth, Kenneth J.; Hand, Arthur R.; Yankova, Maya; Huber, Greg; Lichtman, Jeff W.; Rapoport, Tom A.; Kozlov, Michael M.

    2013-01-01

    The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used novel staining and automated ultra-thin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. PMID:23870120

  4. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs.

    PubMed

    Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W; Schalek, Richard; Hayworth, Kenneth J; Hand, Arthur R; Yankova, Maya; Huber, Greg; Lichtman, Jeff W; Rapoport, Tom A; Kozlov, Michael M

    2013-07-18

    The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.

  5. Endoplasmic Reticulum Calcium, Stress and Cell-to-Cell Adhesion

    PubMed Central

    Mauro, Theodora

    2014-01-01

    Darier's Disease (DD) is caused by mutations in the endoplasmic reticulum (ER) Ca2+ ATPase ATP2A2 (protein SERCA2). Current treatment modalities are ineffective for many patients. This report shows that impaired SERCA2 function, both in DD keratinocytes and in normal keratinocytes treated with the SERCA2-inhibitor thapsigargin, depletes ER Ca2+ stores, leading to constitutive ER stress and increased sensitivity to ER stressors. ER stress, in turn, leads to abnormal cell-to-cell adhesion via impaired redistribution of desmoplakin, desmoglein 3, desmocollin 3 and E-cadherin to the plasma membrane. This report illustrates how ER Ca2+ depletion and the resulting ER stress are central to the pathogenesis of the disease. Additionally, the authors introduce a possible new therapeutic agent, Miglustat. PMID:24924761

  6. Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders.

    PubMed

    Cai, Yu; Arikkath, Jyothi; Yang, Lu; Guo, Ming-Lei; Periyasamy, Palsamy; Buch, Shilpa

    2016-01-01

    The common underlying feature of most neurodegenerative diseases such as Alzheimer disease (AD), prion diseases, Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) involves accumulation of misfolded proteins leading to initiation of endoplasmic reticulum (ER) stress and stimulation of the unfolded protein response (UPR). Additionally, ER stress more recently has been implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. There is evidence that autophagy ameliorates ER stress by eliminating accumulated misfolded proteins. Both abnormal UPR and impaired autophagy have been implicated as a causative mechanism in the development of various neurodegenerative diseases. This review highlights recent advances in the field on the role of ER stress and autophagy in AD, prion diseases, PD, ALS and HAND with the involvement of key signaling pathways in these processes and implications for future development of therapeutic strategies.

  7. Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

    Wang, Pengwei; Hawes, Chris; Hussey, Patrick J

    2017-04-01

    The endoplasmic reticulum (ER) acts as a superhighway with multiple sideroads that connects the different membrane compartments including the ER to the plasma membrane (PM). ER-PM contact sites (EPCSs) are a common feature in eukaryotic organisms, but have not been studied well in plants owing to the lack of molecular markers and to the difficulty in resolving the EPCS structure using conventional microscopy. Recently, however, plant protein complexes required for linking the ER and PM have been identified. This is a further step towards understanding the structure and function of plant EPCSs. We highlight some recent studies in this field and suggest several hypotheses that relate to the possible function of EPCSs in plants.

  8. WLS retrograde transport to the endoplasmic reticulum during Wnt secretion.

    PubMed

    Yu, Jia; Chia, Joanne; Canning, Claire Ann; Jones, C Michael; Bard, Frédéric A; Virshup, David M

    2014-05-12

    Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

  9. Co-chaperones of the mammalian endoplasmic reticulum.

    PubMed

    Melnyk, Armin; Rieger, Heiko; Zimmermann, Richard

    2015-01-01

    In mammalian cells, the rough endoplasmic reticulum or ER plays a central role in the biogenesis of most extracellular plus many organellar proteins and in cellular calcium homeostasis. Therefore, this organelle comprises molecular chaperones that are involved in import, folding/assembly, export, and degradation of polypeptides in millimolar concentrations. In addition, there are calcium channels/pumps and signal transduction components present in the ER membrane that affect and are affected by these processes. The ER lumenal Hsp70, termed immunoglobulin-heavy chain binding protein or BiP, is the central player in all these activities and involves up to seven different co-chaperones, i.e. ER-membrane integrated as well as ER-lumenal Hsp40s, which are termed ERj or ERdj, and two nucleotide exchange factors.

  10. Endoplasmic reticulum stress in diabetes: New insights of clinical relevance.

    PubMed

    Balasubramanyam, Muthuswamy; Lenin, Raji; Monickaraj, Finny

    2010-04-01

    The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. Accumulating evidence suggests that ER stress plays a role in the pathogenesis of diabetes, contributing to pancreatic β-cell loss and insulin resistance. ER stress may also link obesity, inflammation and insulin resistance in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response in diabetes and its complications. Special attention was given to elucidate how ER stress could explain some of the 'clinical paradoxes' such as secondary sulfonylurea failure, initial worsening of retinopathy during tight glycemic control, insulin resistance induced by protease inhibitors and other clinically relevant observations.

  11. Stress Responses from the Endoplasmic Reticulum in Cancer

    PubMed Central

    Kato, Hironori; Nishitoh, Hideki

    2015-01-01

    The endoplasmic reticulum (ER) is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR). The UPR also contributes to the regulation of various intracellular signaling pathways such as calcium signaling and lipid signaling. More recently, the mitochondria-associated ER membrane (MAM), which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signaling, inflammatory signaling, the autophagic response, and the UPR. Interestingly, in cancer, these signaling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signaling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM. PMID:25941664

  12. Assembly of MHC class I molecules within the endoplasmic reticulum.

    PubMed

    Zhang, Yinan; Williams, David B

    2006-01-01

    MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.

  13. Pharmacological Modulators of Endoplasmic Reticulum Stress in Metabolic Diseases

    PubMed Central

    Jung, Tae Woo; Choi, Kyung Mook

    2016-01-01

    The endoplasmic reticulum (ER) is the principal organelle responsible for correct protein folding, a step in protein synthesis that is critical for the functional conformation of proteins. ER stress is a primary feature of secretory cells and is involved in the pathogenesis of numerous human diseases, such as certain neurodegenerative and cardiometabolic disorders. The unfolded protein response (UPR) is a defense mechanism to attenuate ER stress and maintain the homeostasis of the organism. Two major degradation systems, including the proteasome and autophagy, are involved in this defense system. If ER stress overwhelms the capacity of the cell’s defense mechanisms, apoptotic death may result. This review is focused on the various pharmacological modulators that can protect cells from damage induced by ER stress. The possible mechanisms for cytoprotection are also discussed. PMID:26840310

  14. Maternal obesity alters endoplasmic reticulum homeostasis in offspring pancreas.

    PubMed

    Soeda, Jumpei; Mouralidarane, Angelina; Cordero, Paul; Li, Jiawei; Nguyen, Vi; Carter, Rebeca; Kapur, Sabrina R; Pombo, Joaquim; Poston, Lucilla; Taylor, Paul D; Vinciguerra, Manlio; Oben, Jude A

    2016-06-01

    The prevalence of non-alcoholic fatty pancreas disease (NAFPD) is increasing in parallel with obesity rates. Stress-related alterations in endoplasmic reticulum (ER), such as the unfolded protein response (UPR), are associated with obesity. The aim of this study was to investigate ER imbalance in the pancreas of a mice model of adult and perinatal diet-induced obesity. Twenty female C57BL/6J mice were assigned to control (Con) or obesogenic (Ob) diets prior to and during pregnancy and lactation. Their offspring were weaned onto Con or Ob diets up to 6 months post-partum. Then, after sacrifice, plasma biochemical analyses, gene expression, and protein concentrations were measured in pancreata. Offspring of Ob-fed mice had significantly increased body weight (p < 0.001) and plasma leptin (p < 0.001) and decreased insulin (p < 0.01) levels. Maternal obesogenic diet decreased the total and phosphorylated Eif2α and increased spliced X-box binding protein 1 (XBP1). Pancreatic gene expression of downstream regulators of UPR (EDEM, homocysteine-responsive endoplasmic reticulum-resident (HERP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP)) and autophagy-related proteins (LC3BI/LC3BII) were differently disrupted by obesogenic feeding in both mothers and offspring (from p < 0.1 to p < 0.001). Maternal obesity and Ob feeding in their offspring alter UPR in NAFPD, with involvement of proapoptotic and autophagy-related markers. Upstream and downstream regulators of PERK, IRE1α, and ATF6 pathways were affected differently following the obesogenic insults.

  15. Toll-like receptor 4-induced endoplasmic reticulum stress contributes to endothelial dysfunction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Endoplasmic reticulum (ER) stress is implicated as one of the mechanisms for pathophysiology of various cardiometabolic syndromes, including insulin resistance and endothelial dysfunction. ...

  16. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    DTIC Science & Technology

    2016-07-01

    AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...1 July 2015- 30 June 2016 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a...We hypothesize that ER stress induced by glucose in diabetes promotes diabetic CKD through CRT stimulation of TGF-beta-dependent calcium/NFAT

  17. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    DTIC Science & Technology

    2015-07-01

    1 AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...COVERED 07/01/2014-06/30/2015 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a...NUMBER(S) 13. SUPPLEMENTARY NOTES 14. ABSTRACT We hypothesize that ER stress induced by glucose in diabetes promotes diabetic CKD through CRT stimulation

  18. Apoptosis, autophagy & endoplasmic reticulum stress in diabetes mellitus

    PubMed Central

    Demirtas, Levent; Guclu, Aydin; Erdur, Fatih Mehmet; Akbas, Emin Murat; Ozcicek, Adalet; Onk, Didem; Turkmen, Kultigin

    2016-01-01

    The prevalence of diabetes mellitus (DM) is increasing secondary to increased consumption of food and decreased physical activity worldwide. Hyperglycaemia, insulin resistance and hypertrophy of pancreatic beta cells occur in the early phase of diabetes. However, with the progression of diabetes, dysfunction and loss of beta cells occur in both types 1 and 2 DM. Programmed cell death also named apoptosis is found to be associated with diabetes, and apoptosis of beta cells might be the main mechanism of relative insulin deficiency in DM. Autophagic cell death and apoptosis are not entirely distinct programmed cell death mechanisms and share many of the regulator proteins. These processes can occur in both physiologic and pathologic conditions including DM. Besides these two important pathways, endoplasmic reticulum (ER) also acts as a cell sensor to monitor and maintain cellular homeostasis. ER stress has been found to be associated with autophagy and apoptosis. This review was aimed to describe the interactions between apoptosis, autophagy and ER stress pathways in DM. PMID:28256459

  19. Chlorpyrifos induces endoplasmic reticulum stress in JEG-3 cells.

    PubMed

    Reyna, Luciana; Flores-Martín, Jésica; Ridano, Magali E; Panzetta-Dutari, Graciela M; Genti-Raimondi, Susana

    2017-04-01

    Chlorpyrifos (CPF) is an organophosphorous pesticide widely used in agricultural, industrial, and household applications. We have previously shown that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway. Considering that there is a relationship between oxidative stress and endoplasmic reticulum stress (ER), herein we investigated whether CPF also induces ER stress in JEG-3 cells. Cells were exposed to 50μM or 100μM CPF during 24h in conditions where cell viability was not altered. Western blot and PCR assays were used to explore the protein and mRNA levels of ER stress biomarkers, respectively. CPF induced an increase of the typical ER stress-related proteins, such as GRP78/BiP and IRE1α, a sensor for the unfolded protein response, as well as in phospho-eIF2α and XBP1 mRNA splicing. Additionally, CPF led to a decrease in p53 protein expression. The downregulation of p53 levels induced by CPF was partially blocked when cells were exposed to CPF in the presence of the proteasome inhibitor MG132. Altogether, these findings point out that CPF induces ER stress in JEG-3 cells; however these cells are able to attenuate it downregulating the levels of the pro-apoptotic protein p53.

  20. Endoplasmic Reticulum Stress Interacts With Inflammation in Human Diseases.

    PubMed

    Cao, Stewart Siyan; Luo, Katherine L; Shi, Lynn

    2016-02-01

    The endoplasmic reticulum (ER) is a critical organelle for normal cell function and homeostasis. Disturbance in the protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro-apoptotic pathways depending on the type of insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, infectious, neurodegenerative, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways have been shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains poorly understood. Further understanding of the biochemistry, cell biology, and physiology may enable the development of novel therapies that spontaneously target these pathogenic pathways.

  1. Protein quality control, retention, and degradation at the endoplasmic reticulum.

    PubMed

    Benyair, Ron; Ron, Efrat; Lederkremer, Gerardo Z

    2011-01-01

    In order to maintain proper cellular functions, all living cells, from bacteria to mammalian cells, must carry out a rigorous quality control process in which nascent and newly synthesized proteins are examined. An important role of this process is to protect cells against pathological accumulation of unfolded and misfolded proteins. The endoplasmic reticulum (ER) has evolved as a staging ground for secretory protein synthesis with distinct sites for entry, quality control, and exit. In the ER, most proteins are N-glycosylated, a posttranslational modification that defines the quality control pathway that the protein will undergo. The folding state of glycoproteins is revealed by specific modifications of their N-glycans. Regardless of size and posttranslational modifications, the folding states of all proteins must be identified as unfolded, properly folded, or terminally misfolded and accordingly subjected to ER retention and continued folding attempts, export and maturation, or retrotranslocation to the cytosol for degradation. These processes involve specialized machineries that utilize molecular chaperones, protein- and N-glycan-modifying enzymes, and lectins for protein folding and quality control and ubiquitination and degradation machineries for disposal. All these machineries are regulated by a signaling pathway, the unfolded protein response, which upregulates ER functions when under the stress of high protein load. Here, we describe the molecular mechanisms that are implicated and discuss recent data that underline the importance of compartmentalization in the segregation of the various functions of the ER for their correct function.

  2. Proteostasis: bad news and good news from the endoplasmic reticulum.

    PubMed

    Noack, Julia; Brambilla Pisoni, Giorgia; Molinari, Maurizio

    2014-01-01

    The endoplasmic reticulum (ER) is an intracellular compartment dedicated to the synthesis and maturation of secretory and membrane proteins, totalling about 30% of the total eukaryotic cells proteome. The capacity to produce correctly folded polypeptides and to transport them to their correct intra- or extracellular destinations relies on proteostasis networks that regulate and balance the activity of protein folding, quality control, transport and degradation machineries. Nutrient and environmental changes, pathogen infection aging and, more relevant for the topics discussed in this review, mutations that impair attainment of the correct 3D structure of nascent polypeptide chains may compromise the activity of the proteostasis networks with devastating consequences on cells, organs and organisms' homeostasis. Here we present a review of mechanisms regulating folding and quality control of proteins expressed in the ER, and we describe the protein degradation and the ER stress pathways activated by the expression of misfolded proteins in the ER lumen. Finally, we highlight select examples of proteopathies (also known as conformational disorders or protein misfolding diseases) caused by protein misfolding in the ER and/or affecting cellular proteostasis and therapeutic interventions that might alleviate or cure the disease symptoms.

  3. Endoplasmic reticulum stress response in yeast and humans

    PubMed Central

    Wu, Haoxi; Ng, Benjamin S. H.; Thibault, Guillaume

    2014-01-01

    Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases. PMID:24909749

  4. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

    PubMed Central

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J.; Kohalmi, Susanne E.; Menassa, Rima

    2016-01-01

    Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles. PMID:27242885

  5. Small GTPases and Brucella entry into the endoplasmic reticulum.

    PubMed

    de Bolle, Xavier; Letesson, Jean-Jacques; Gorvel, Jean-Pierre

    2012-12-01

    A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.

  6. The Role of the Endoplasmic Reticulum in Peroxisome Biogenesis

    PubMed Central

    Dimitrov, Lazar; Lam, Sheung Kwan; Schekman, Randy

    2013-01-01

    Peroxisomes are essential cellular organelles involved in lipid metabolism. Patients affected by severe peroxisome biogenesis disorders rarely survive their first year. Genetic screens in several model organisms have identified more than 30 PEX genes that are required for the formation of functional peroxisomes. Despite significant work on the PEX genes, the biogenic origin of peroxisomes remains controversial. For at least two decades, the prevailing model postulated that peroxisomes propagate by growth and fission of preexisting peroxisomes. In this review, we focus on the recent evidence supporting a new, semiautonomous model of peroxisomal biogenesis. According to this model, peroxisomal membrane proteins (PMPs) traffic from the endoplasmic reticulum (ER) to the peroxisome by a vesicular budding, targeting, and fusion process while peroxisomal matrix proteins are imported into the organelle by an autonomous, posttranslational mechanism. We highlight the contradictory conclusions reached to answer the question of how PMPs are inserted into the ER. We then review what we know and what still remains to be elucidated about the mechanism of PMP exit from the ER and the contribution of preperoxisomal vesicles to mature peroxisomes. Finally, we discuss discrepancies in our understanding of de novo peroxisome biogenesis in wild-type cells. We anticipate that resolving these key issues will lead to a more complete picture of peroxisome biogenesis. PMID:23637287

  7. Microtubules and the endoplasmic reticulum are highly interdependent structures

    PubMed Central

    1986-01-01

    The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system. PMID:3533956

  8. Apoptosis, autophagy & endoplasmic reticulum stress in diabetes mellitus.

    PubMed

    Demirtas, Levent; Guclu, Aydin; Erdur, Fatih Mehmet; Akbas, Emin Murat; Ozcicek, Adalet; Onk, Didem; Turkmen, Kultigin

    2016-10-01

    The prevalence of diabetes mellitus (DM) is increasing secondary to increased consumption of food and decreased physical activity worldwide. Hyperglycaemia, insulin resistance and hypertrophy of pancreatic beta cells occur in the early phase of diabetes. However, with the progression of diabetes, dysfunction and loss of beta cells occur in both types 1 and 2 DM. Programmed cell death also named apoptosis is found to be associated with diabetes, and apoptosis of beta cells might be the main mechanism of relative insulin deficiency in DM. Autophagic cell death and apoptosis are not entirely distinct programmed cell death mechanisms and share many of the regulator proteins. These processes can occur in both physiologic and pathologic conditions including DM. Besides these two important pathways, endoplasmic reticulum (ER) also acts as a cell sensor to monitor and maintain cellular homeostasis. ER stress has been found to be associated with autophagy and apoptosis. This review was aimed to describe the interactions between apoptosis, autophagy and ER stress pathways in DM.

  9. Arachidonoyl-Specific Diacylglycerol Kinase ε and the Endoplasmic Reticulum

    PubMed Central

    Nakano, Tomoyuki; Matsui, Hirooki; Tanaka, Toshiaki; Hozumi, Yasukazu; Iseki, Ken; Kawamae, Kaneyuki; Goto, Kaoru

    2016-01-01

    The endoplasmic reticulum (ER) comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA) serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK) that phosphorylates diacylglycerol (DG) to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology, and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER. PMID:27917381

  10. Coordination of Endoplasmic Reticulum (ER) Signaling During Maize Seed Development

    SciTech Connect

    Boston, Rebecca S.

    2010-11-20

    Seed storage reserves represent one of the most important sources of renewable fixed carbon and nitrogen found in nature. Seeds are well-adapted for diverting metabolic resources to synthesize storage proteins as well as enzymes and structural proteins needed for their transport and packaging into membrane bound storage protein bodies. Our underlying hypothesis is that the endoplasmic reticulum (ER) stress response provides the critical cellular control of metabolic flux required for optimal accumulation of storage reserves in seeds. This highly conserved response is a cellular mechanism to monitor the protein folding environment of the ER and restore homeostasis in the presence of unfolded or misfolded proteins. In seeds, deposition of storage proteins in protein bodies is a highly specialized process that takes place even in the presence of mutant proteins that no longer fold and package properly. The capacity of the ER to deposit these aberrant proteins in protein bodies during a period that extends several weeks provides an excellent model for deconvoluting the ER stress response of plants. We have focused in this project on the means by which the ER senses and responds to functional perturbations and the underlying intracellular communication that occurs among biosynthetic, trafficking and degradative pathways for proteins during seed development.

  11. Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum.

    PubMed

    Missiroli, Sonia; Morganti, Claudia; Giorgi, Carlotta; Pinton, Paolo

    2016-01-01

    Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.

  12. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    PubMed

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  13. Association of Legionella pneumophila with the macrophage endoplasmic reticulum.

    PubMed Central

    Swanson, M S; Isberg, R R

    1995-01-01

    Legionella pneumophila replicates within a membrane-bounded compartment that is studded with ribosomes. In this study we investigated whether these ribosomes originate from the cytoplasmic pool or are associated with host endoplasmic reticulum (ER). Immunofluorescence and electron microscopic localization studies of ER proteins in macrophages infected with L. pneumophila indicated that the bacteria reside in a compartment surrounded by ER. An L. pneumophila mutant that grows slowly in macrophages was slow to associate with host ER, providing genetic evidence in support of the hypothesis that this specialized vacuole is required for intracellular bacterial growth. Ultrastructural studies, in which the ER luminal protein BiP was labeled by immunoperoxidase cytochemistry, revealed that L. pneumophila replication vacuoles resemble nascent autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagy, increased association of the bacteria with the ER and enhanced bacterial growth. These results are compatible with the hypothesis that L. pneumophila exploits the autophagy machinery of macrophages to establish an intracellular niche favorable for replication. PMID:7642298

  14. Regulation of endoplasmic reticulum turnover by selective autophagy.

    PubMed

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-06-18

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

  15. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    PubMed

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

  16. Hydrogen sulfide, endoplasmic reticulum stress and alcohol mediated neurotoxicity.

    PubMed

    George, Akash K; Behera, Jyotirmaya; Kelly, Kimberly E; Zhai, Yuankun; Tyagi, Neetu

    2017-02-14

    Alcohol is one of the most socially accepted addictive drugs in modern society. Its abuse affects virtually all organ systems with the central nervous system (CNS) being particularly vulnerable to excessive alcohol exposure. Alcohol exposure also causes profound damage to both the adult and developing brain. Excessive alcohol consumption induces numerous pathophysiological stress responses, one of which is the endoplasmic reticulum (ER) stress response. Potential mechanisms that trigger the alcohol induced ER stress response are either directly or indirectly related to alcohol metabolism, which include toxic levels of acetaldehyde and homocysteine, oxidative stress and abnormal epigenetic modifications. Growing evidence suggests that H2S is the most recently recognized gasotransmitter with tremendous physiological protective functions against oxidative stress induced neurotoxicity. In this review we address the alcohol induced oxidative stress mediated ER stress and the role of H2S in its mitigation in the context of alcohol neurotoxicity. Interruption of ER stress triggers is anticipated to have therapeutic benefits for alcohol mediated diseases and disorders.

  17. Heme oxygenase-1 comes back to endoplasmic reticulum

    SciTech Connect

    Kim, Hong Pyo; Pae, Hyun-Ock; Back, Sung Hun; Chung, Su Wol; Woo, Je Moon; Son, Yong; Chung, Hun-Taeg

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  18. Terasaki Ramps in the Endoplasmic Reticulum: Structure, Function and Formation

    NASA Astrophysics Data System (ADS)

    Huber, Greg; Guven, Jemal; Valencia, Dulce-Maria

    2015-03-01

    The endoplasmic reticulum (ER) has long been considered an exceedingly important and complex cellular organelle in eukaryotes (like you). It is a membrane structure, part folded lamellae, part tubular network, that both envelopes the nucleus and threads its way outward, all the way to the cell's periphery. Despite the elegant mechanics of bilayer membranes offered by the work of Helfrich and Canham, as far as the ER is concerned, theory has mostly sat on the sidelines. However, refined imaging of the ER has recently revealed beautiful and subtle geometrical forms - simple geometries, from the mathematical point of view - which some have called a ``parking garage for ribosomes.'' I'll review the discovery and physics of Terasaki ramps and discuss their relation to cell-biological questions, such as ER and nuclear-membrane re-organization during mitosis. Rather than being a footnote in a textbook on differential geometry, these structures suggest answers to a number of the ER's structure-function problems.

  19. A Molecular Web: Endoplasmic Reticulum Stress, Inflammation, and Oxidative Stress

    PubMed Central

    Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d’Hellencourt, Christian; Ravanan, Palaniyandi

    2014-01-01

    Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress. PMID:25120434

  20. PROTEOMICS ANALYSIS OF ROUGH ENDOPLASMIC RETICULUM IN PANCREATIC BETA CELLS

    PubMed Central

    Lee, Jin-sook; Wu, Yanning; Skallos, Patracia; Fang, Jingye; Zhang, Xuebao; Karnovsky, Alla; Woods, James; Stemmer, Paul M.; Liu, Ming; Zhang, Kezhong; Chen, Xuequn

    2015-01-01

    Pancreatic beta cells have well-developed endoplasmic reticulum (ER) to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by one dimensional SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. Gene ontology analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. PMID:25546123

  1. The Mammalian Endoplasmic Reticulum-Associated Degradation System

    PubMed Central

    Olzmann, James A.; Kopito, Ron R.; Christianson, John C.

    2013-01-01

    The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells. PMID:23232094

  2. PERK-opathies: An Endoplasmic Reticulum Stress Mechanism Underlying Neurodegeneration.

    PubMed

    Bell, Michelle C; Meier, Shelby E; Ingram, Alexandria L; Abisambra, Jose F

    2016-01-01

    The unfolded protein response (UPR) plays a vital role in maintaining cell homeostasis as a consequence of endoplasmic reticulum (ER) stress. However, prolonged UPR activity leads to cell death. This time-dependent dual functionality of the UPR represents the adaptive and cytotoxic pathways that result from ER stress. Chronic UPR activation in systemic and neurodegenerative diseases has been identified as an early sign of cellular dyshomeostasis. The Protein Kinase R-like ER Kinase (PERK) pathway is one of three major branches in the UPR, and it is the only one to modulate protein synthesis as an adaptive response. The specific identification of prolonged PERK activity has been correlated with the progression of disorders such as diabetes, Alzheimer's disease, and cancer, suggesting that PERK plays a role in the pathology of these disorders. For the first time, the term "PERK-opathies" is used to group these diseases in which PERK mediates detriment to the cell culminating in chronic disorders. This article reviews the literature documenting links between systemic disorders with the UPR, but with a specific emphasis on the PERK pathway. Then, articles reporting links between the UPR, and more specifically PERK, and neurodegenerative disorders are presented. Finally, a therapeutic perspective is discussed, where PERK interventions could be potential remedies for cellular dysfunction in chronic neurodegenerative disorders.

  3. Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

    PubMed

    Hong, Seong-Ho; Chang, Seung-Hee; Cho, Kyung-Cho; Kim, Sanghwa; Park, Sungjin; Lee, Ah Young; Jiang, Hu-Lin; Kim, Hyeon-Jeong; Lee, Somin; Yu, Kyeong-Nam; Seo, Hwi Won; Chae, Chanhee; Kim, Kwang Pyo; Park, Jongsun; Cho, Myung-Haing

    2016-10-04

    Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies.

  4. Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy

    PubMed Central

    Hong, Seong-Ho; Chang, Seung-Hee; Cho, Kyung-Cho; Kim, Sanghwa; Park, Sungjin; Lee, Ah Young; Jiang, Hu-Lin; Kim, Hyeon-Jeong; Lee, Somin; Yu, Kyeong-Nam; Seo, Hwi Won; Chae, Chanhee; Kim, Kwang Pyo; Park, Jongsun; Cho, Myung-Haing

    2016-01-01

    Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies. PMID:27588471

  5. CDIP1-BAP31 complex transduces apoptotic signals from endoplasmic reticulum to mitochondria under endoplasmic reticulum stress.

    PubMed

    Namba, Takushi; Tian, Fang; Chu, Kiki; Hwang, So-Young; Yoon, Kyoung Wan; Byun, Sanguine; Hiraki, Masatsugu; Mandinova, Anna; Lee, Sam W

    2013-10-31

    Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

  6. Obesity-induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

    PubMed

    Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

    2017-03-09

    Obesity is a significant risk factor for the acute respiratory distress syndrome (ARDS). The mechanisms underlying this association are unknown. We recently showed that diet-induced obese (DIO) mice exhibit pulmonary vascular endothelial dysfunction which is associated with enhanced susceptibility to lipopolysaccharide (LPS)-induced lung injury. Here, we demonstrate that lung endothelial dysfunction in DIO mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins including PERK, IREα and ATF6, in whole lung and in lung endothelial cells isolated from DIO mice. Further, we found that lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of DIO mice. Similar changes were observed in lung endothelial cells and in whole lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation; indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-PBA, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in DIO mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the endoplasmic reticulum of pulmonary endothelial cells might protect against ARDS in obese individuals.

  7. Sertraline induces endoplasmic reticulum stress in hepatic cells.

    PubMed

    Chen, Si; Xuan, Jiekun; Couch, Letha; Iyer, Advait; Wu, Yuanfeng; Li, Quan-Zhen; Guo, Lei

    2014-08-01

    Sertraline is used for the treatment of depression, and is also used for the treatment of panic, obsessive-compulsive, and post-traumatic stress disorders. Previously, we have demonstrated that sertraline caused hepatic cytotoxicity, with mitochondrial dysfunction and apoptosis being underlying mechanisms. In this study, we used microarray and other biochemical and molecular analyses to identify endoplasmic reticulum (ER) stress as a novel molecular mechanism. HepG2 cells were exposed to sertraline and subjected to whole genome gene expression microarray analysis. Pathway analysis revealed that ER stress is among the significantly affected biological changes. We confirmed the increased expression of ER stress makers by real-time PCR and Western blots. The expression of typical ER stress markers such as PERK, IRE1α, and CHOP was significantly increased. To study better ER stress-mediated drug-induced liver toxicity; we established in vitro systems for monitoring ER stress quantitatively and efficiently, using Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) as ER stress reporters. These in vitro systems were validated using well-known ER stress inducers. In these two reporter assays, sertraline inhibited the secretion of Gluc and SEAP. Moreover, we demonstrated that sertraline-induced apoptosis was coupled to ER stress and that the apoptotic effect was attenuated by 4-phenylbutyrate, a potent ER stress inhibitor. In addition, we showed that the MAP4K4-JNK signaling pathway contributed to the process of sertraline-induced ER stress. In summary, we demonstrated that ER stress is a mechanism of sertraline-induced liver toxicity.

  8. Endoplasmic Reticulum Stress Is Chronically Activated in Chronic Pancreatitis*

    PubMed Central

    Sah, Raghuwansh P.; Garg, Sushil K.; Dixit, Ajay K.; Dudeja, Vikas; Dawra, Rajinder K.; Saluja, Ashok K.

    2014-01-01

    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T−/−), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T−/− mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies. PMID:25077966

  9. Endoplasmic reticulum stress is chronically activated in chronic pancreatitis.

    PubMed

    Sah, Raghuwansh P; Garg, Sushil K; Dixit, Ajay K; Dudeja, Vikas; Dawra, Rajinder K; Saluja, Ashok K

    2014-10-03

    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T(-/-)), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T(-/-) mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.

  10. Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

    PubMed Central

    Chalat, Madhavan; Menon, Indu; Turan, Zeynep; Menon, Anant K.

    2012-01-01

    Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t½ < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)hexanoyl)-d-glucosyl-β1–1′-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity. PMID:22427661

  11. Endoplasmic reticulum stress activation during total knee arthroplasty

    PubMed Central

    Hocker, Austin D; Boileau, Ryan M; Lantz, Brick A; Jewett, Brian A; Gilbert, Jeffrey S; Dreyer, Hans C

    2013-01-01

    Total knee arthroplasty (TKA) is the most common remediation for knee pain from osteoarthritis (OA) and is performed 650,000 annually in the U.S. A tourniquet is commonly used during TKA which causes ischemia and reperfusion (I/R) to the lower limb but the effects of I/R on muscle are not fully understood. Previous reports suggest upregulation of cell stress and catabolism and downregulation of markers of cap-dependent translation during and after TKA. I/R has also been shown to cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). We hypothesized that the UPR would be activated in response to ER stress during TKA. We obtained muscle biopsies from the vastus lateralis at baseline, before TKA; at maximal ischemia, prior to tourniquet deflation; and during reperfusion in the operating room. Phosphorylation of 4E-BP1 and AKT decreased during ischemia (−28%, P < 0.05; −20%, P < 0.05, respectively) along with an increase in eIF2α phosphorylation (64%, P < 0.05) suggesting decreased translation initiation. Cleaved ATF6 protein increased in ischemia (39%, P = 0.056) but returned to baseline during reperfusion. CASP3 activation increased during reperfusion compared to baseline (23%, P < 0.05). XBP1 splicing assays revealed an increase in spliced transcript during ischemia (31%, P < 0.05) which diminished during reperfusion. These results suggest that in response to I/R during TKA all three branches of the ER stress response are activated. PMID:24159375

  12. Endoplasmic reticulum stress: key promoter of rosacea pathogenesis.

    PubMed

    Melnik, Bodo C

    2014-12-01

    Recent scientific interest in the pathogenesis of rosacea focuses on abnormally high facial skin levels of cathelicidin and the trypsin-like serine protease kallikrein 5 (KLK5) that cleaves the cathelicidin precursor protein into the bioactive fragment LL-37, which exerts crucial proinflammatory, angiogenic and antimicrobial activities. Furthermore, increased expression of toll-like receptor 2 (TLR2) has been identified in rosacea skin supporting the participation of the innate immune system. Notably, TLRs are expressed on sensory neurons and increase neuronal excitability linking TLR signalling to the transmission of neuroinflammatory responses. It is the intention of this viewpoint to present a unifying concept that links all known clinical trigger factors of rosacea such as UV irradiation, heat, skin irritants and special foods to one converging point: enhanced endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR). ER stress via upregulation of transcription factor ATF4 increases TLR2 expression, resulting in enhanced production of cathelicidin and KLK5 mediating downstream proinflammatory, angiogenic and antimicrobial signalling. The presented concept identifies rosacea trigger factors as environmental stressors that enhance the skin's ER stress response. Exaggerated cutaneous ER stress that stimulates the TLR2-driven inflammatory response may involve sebocytes, keratinocytes, monocyte-macrophages and sensory cutaneous neurons. Finally, all antirosacea drugs are proposed to attenuate the ER stress signalling cascade at some point. Overstimulated ER stress signalling may have evolutionarily evolved as a compensatory mechanism to balance impaired vitamin D-driven LL-37-mediated antimicrobial defenses due to lower exposure of UV-B irradiation of the northern Celtic population.

  13. Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics

    PubMed Central

    Kaufman, Randal J.; Malhotra, Jyoti D.

    2014-01-01

    Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. “Cellular calcium overload” is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associate changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis [4] [5] [6] [7]. Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered [8] [9] [10]. The next questions are how they are regulated for exquisite tight control of ER – mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. PMID:24690484

  14. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein*

    PubMed Central

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S.; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M.; Brandes, Ralf P.

    2016-01-01

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum. PMID:26861875

  15. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.

    PubMed

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M; Brandes, Ralf P

    2016-03-25

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.

  16. Autophagy decreases alveolar macrophage apoptosis by attenuating endoplasmic reticulum stress and oxidative stress

    PubMed Central

    Fan, Tao; Chen, Lei; Huang, Zhixin; Mao, Zhangfan; Wang, Wei; Zhang, Boyou; Xu, Yao; Pan, Shize; Hu, Hao; Geng, Qing

    2016-01-01

    To study the impact of autophagy on alveolar macrophage apoptosis and its mechanism in the early stages of hypoxia, we established a cell hypoxia-reoxygenation model and orthotopic left lung ischemia-reperfusion model. Rat alveolar macrophages stably expressing RFP-LC3 were treated with autophagy inhibitor (3-methyladenine, 3-MA) or autophagy promoter (rapamycin), followed by hypoxia-reoxygenation treatment 2 h, 4 h or 6 h later. Twenty Sprague-Dawley male rats were randomly divided into four different groups: no blocking of left lung hilum (model group), left lung hilum blocked for 1h with DMSO lavage (control group), left lung hilum blocked for 1 h with 100 ml/kg 3-MA (5 μmol/L) lavage (3-MA group), and left lung hilum blocked for 1 h with 100 ml/kg rapamycin (250 nmol/L) lavage (rapamycin group). Rapamycin decreased the unfolded protein response, which reduced endoplasmic reticulum stress-mediated apoptosis in the presence of oxygen deficiency. Rapamycin increased superoxide dismutase activities and decreased malondialdehyde levels, whereas 3-MA decreased superoxide dismutase activities and increased malondialdehyde levels. Thus, autophagy decreases alveolar macrophage apoptosis by attenuating endoplasmic reticulum stress and oxidative stress in the early stage of hypoxia in vitro and in vivo. This could represent a new approach to protecting against lung ischemia-reperfusion injury. PMID:27888631

  17. Selenoprotein K modulate intracellular free Ca(2+) by regulating expression of calcium homoeostasis endoplasmic reticulum protein.

    PubMed

    Wang, Chao; Li, Ruimin; Huang, Yalan; Wang, Miao; Yang, Fan; Huang, Dana; Wu, Chunli; Li, Yue; Tang, Yijun; Zhang, Renli; Cheng, Jinquan

    2017-03-18

    Selenoprotein K (SelK) is an 11-kDa selenoprotein, which may be involved in the regulation of oxidative stress, endoplasmic reticulum (ER) stress and immune response. To explore the function of SelK in the process of immune response, several short-hairpin RNAs (shRNA) were designed for the construction of recombinant plasmids to down-regulate the expression of SelK gene in vitro. These shRNAs specifically and efficiently interfered with the expression of SelK at both mRNA and protein levels. The expression of calcium homoeostasis endoplasmic reticulum protein (CHERP) and the intracellular free Ca(2+) concentration were significantly down-regulated in anti-CD3 stimulated SelK-knockdown cells. The expression of Interleukin 2 receptor alpha chain (IL-2Rα) and the secretion of Interleukin 4 (IL-4), which play a significant role in the process of T cell activation and proliferation, were also reduced in SelK-knockdown cells. Selenomethionine (Se-Met) at an optimum concentration of 5 μM could up-regulate SelK expression and reverse the change of the expression of CHERP and the intracellular free calcium caused by SelK-knockdown. These results hereby imply SelK may regulate the release of Ca(2+) by CHERP and play an important role in the proliferation and differentiation of T cell by TCR stimulation.

  18. Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells

    NASA Technical Reports Server (NTRS)

    Zheng, H. Q.; Staehelin, L. A.

    2001-01-01

    The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.

  19. Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis

    PubMed Central

    Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo

    2016-01-01

    Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. PMID:27999379

  20. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

    PubMed

    Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

    2014-10-01

    Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure.

  1. Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology.

    PubMed

    Guzel, Elif; Arlier, Sefa; Guzeloglu-Kayisli, Ozlem; Tabak, Mehmet Selcuk; Ekiz, Tugba; Semerci, Nihan; Larsen, Kellie; Schatz, Frederick; Lockwood, Charles Joseph; Kayisli, Umit Ali

    2017-04-08

    The endoplasmic reticulum (ER), comprises 60% of the total cell membrane and interacts directly or indirectly with several cell organelles i.e., Golgi bodies, mitochondria and proteasomes. The ER is usually associated with large numbers of attached ribosomes. During evolution, ER developed as the specific cellular site of synthesis, folding, modification and trafficking of secretory and cell-surface proteins. The ER is also the major intracellular calcium storage compartment that maintains cellular calcium homeostasis. During the production of functionally effective proteins, several ER-specific molecular steps sense quantity and quality of synthesized proteins as well as proper folding into their native structures. During this process, excess accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress, the homeostatic coping mechanism that activates an ER-specific adaptation program, (the unfolded protein response; UPR) to increase ER-associated degradation of structurally and/or functionally defective proteins, thus sustaining ER homeostasis. Impaired ER homeostasis results in aberrant cellular responses, contributing to the pathogenesis of various diseases. Both female and male reproductive tissues undergo highly dynamic cellular, molecular and genetic changes such as oogenesis and spermatogenesis starting in prenatal life, mainly controlled by sex-steroids but also cytokines and growth factors throughout reproductive life. These reproductive changes require ER to provide extensive protein synthesis, folding, maturation and then their trafficking to appropriate cellular location as well as destroying unfolded/misfolded proteins via activating ER-associated degradation mediated proteasomes. Many studies have now shown roles for ER stress/UPR signaling cascades in the endometrial menstrual cycle, ovarian folliculogenesis and oocyte maturation, spermatogenesis, fertilization, pre-implantation embryo development and pregnancy and parturition

  2. Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino acids.

    PubMed

    Uchio, Naohiro; Oma, Yoko; Toriumi, Kazuya; Sasagawa, Noboru; Tanida, Isei; Fujita, Eriko; Kouroku, Yoriko; Kuroda, Reiko; Momoi, Takashi; Ishiura, Shoichi

    2007-11-01

    Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.

  3. Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

    PubMed Central

    Iturriaga, G; Jefferson, R A; Bevan, M W

    1989-01-01

    The correct compartmentation of proteins to the endomembrane system, mitochondria, or chloroplasts requires an amino-terminal signal peptide. The major tuber protein of potato, patatin, has a signal peptide in common with many other plant storage proteins. When the putative signal peptide of patatin was fused to the bacterial reporter protein beta-glucuronidase, the fusion proteins were translocated to the endoplasmic reticulum in planta and in vitro. In addition, translocated beta-glucuronidase was modified by glycosylation, and the signal peptide was correctly processed. In the presence of an inhibitor of glycosylation, tunicamycin, the enzymatically active form of beta-glucuronidase was assembled in the endoplasmic reticulum. This is the first report of targeting a cytoplasmic protein to the endoplasmic reticulum of plants using a signal peptide. PMID:2535509

  4. Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress

    PubMed Central

    Hosoi, Toru; Yamaguchi, Rie; Noji, Kikuko; Matsuo, Suguru; Baba, Sachiko; Toyoda, Keisuke; Suezawa, Takahiro; Kayano, Takaaki; Tanaka, Shinpei; Ozawa, Koichiro

    2014-01-01

    Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress-induced leptin resistance, characterized by insensitivity to the actions of the anti-obesity hormone leptin. This result was further supported by flurbiprofen attenuating high-fat diet-induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti-obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity. Subject Categories Metabolism; Pharmacology & Drug Discovery PMID:24421337

  5. Unusual configurations of endoplasmic reticulum in cells of acute promyelocytic leukemia.

    PubMed

    Parkin, J L; Brunning, R D

    1978-08-01

    An ultrastructural study of leukemia cells from 8 patients with acute promyelocytic leukemia revealed several features that have not previously been emphasized: prominent dilated rough endoplasmic reticulum and two unusual configurations of endoplasmic reticulum (ER). The two membrane structures, multilaminar ER and complex stellate arrangements of ER, appeared to be morphogenetically related. The multilaminar ER was observed in every mitotic cell and less frequently in interphase cells. The stellate ER complex was observed only in interphase cells. Ultrastructural evidence is presented to support the possible evolution of the stellate ER complex from the multilaminar ER.

  6. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  7. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

  8. Mutations in the SPTLC1 protein cause mitochondrial structural abnormalities and endoplasmic reticulum stress in lymphoblasts.

    PubMed

    Myers, Simon J; Malladi, Chandra S; Hyland, Ryan A; Bautista, Tara; Boadle, Ross; Robinson, Phillip J; Nicholson, Garth A

    2014-07-01

    Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.

  9. Microtubules as key coordinators of nuclear envelope and endoplasmic reticulum dynamics during mitosis.

    PubMed

    Schlaitz, Anne-Lore

    2014-07-01

    During mitosis, cells comprehensively restructure their interior to promote the faithful inheritance of DNA and cytoplasmic contents. In metazoans, this restructuring entails disassembly of the nuclear envelope, redistribution of its components into the endoplasmic reticulum (ER) and eventually nuclear envelope reassembly around the segregated chromosomes. The microtubule cytoskeleton has recently emerged as a critical regulator of mitotic nuclear envelope and ER dynamics. Microtubules and associated molecular motors tear open the nuclear envelope in prophase and remove nuclear envelope remnants from chromatin. Additionally, two distinct mechanisms of microtubule-based regulation of ER dynamics operate later in mitosis. First, association of the ER with microtubules is reduced, preventing invasion of ER into the spindle area, and second, organelle membrane is actively cleared from metaphase chromosomes. However, we are only beginning to understand the role of microtubules in shaping and distributing ER and other organelles during mitosis.

  10. Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling.

    PubMed

    Carrara, Marta; Prischi, Filippo; Nowak, Piotr R; Ali, Maruf Mu

    2015-06-03

    Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein-folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X-ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.

  11. Aging induced endoplasmic reticulum stress alters sleep and sleep homeostasis.

    PubMed

    Brown, Marishka K; Chan, May T; Zimmerman, John E; Pack, Allan I; Jackson, Nicholas E; Naidoo, Nirinjini

    2014-06-01

    Alterations in the quality, quantity, and architecture of baseline and recovery sleep have been shown to occur during aging. Sleep deprivation induces endoplasmic reticular (ER) stress and upregulates a protective signaling pathway termed the unfolded protein response. The effectiveness of the adaptive unfolded protein response is diminished by age. Previously, we showed that endogenous chaperone levels altered recovery sleep in Drosophila melanogaster. We now report that acute administration of the chemical chaperone sodium 4-phenylbutyrate (PBA) reduces ER stress and ameliorates age-associated sleep changes in Drosophila. PBA consolidates both baseline and recovery sleep in aging flies. The behavioral modifications of PBA are linked to its suppression of ER stress. PBA decreased splicing of X-box binding protein 1 and upregulation of phosphorylated elongation initiation factor 2 α, in flies that were subjected to sleep deprivation. We also demonstrate that directly activating ER stress in young flies fragments baseline sleep and alters recovery sleep. Alleviating prolonged or sustained ER stress during aging contributes to sleep consolidation and improves recovery sleep or sleep debt discharge.

  12. Changes in cellular infrastructure after induced endoplasmic reticulum stress in Moniliophthora perniciosa.

    PubMed

    Basso, Tatiana Setenta; Vita-Santos, Evelyn; Marisco, Gabriele; Pungartnik, Cristina; Brendel, Martin

    2016-09-01

    Moniliophthora perniciosa is a basidiomycete fungus that causes witches' broom disease in Theobroma cacao We analyzed the morphology and survival of fungal hyphae and endoplasmic reticulum (ER) remodeling in either glucose- or glycerol-grown M. perniciosa after treatment with ER stress-inducing chemicals dithiothreitol (DTT) or tunicamycin (TM). Changes in intracellular redox potential can cause endoplasmic reticulum (ER) stress due to diminished efficiency in protein folding that could in turn reduce cell survival. Such stress diminishes protein-folding efficiency that could in turn reduce cell survival. Light microscopy revealed morphological changes in hyphae after TM but not after DTT treatment, regardless of the media carbon source. Decrease in fungal survival, after both TM and DTT treatments, was dose-dependent and glycerol-grown cells showed a higher resistance to both chemicals compared to glucose-grown cells. Electron microscopy showed TM and DDT-induced ER stress in M. perniciosa as evidenced by structural alterations of the organelle. The volume of ER structures increased as a typical consequence of unfolded protein stress, and the number of autophagosomes was higher. In glycerol-grown fungus DTT treatment slightly induced expression of molecular chaperone BiP. The TM exposure-induced expression of gene MpIRE1, involved in signaling of the unfolded protein response, was higher in glycerol than glucose-grown cells. Such difference was not observable with expression of gene MpATG8, encoding a key protein in autosome formation, that was induced 1.4-fold and 1.2-fold in glucose or glycerol-grown cells, respectively. DHE-based fluorescence assay showed M. perniciosa oxidative stress induced by H2O2, and treated cells had a higher level of oxidative stress compared to control. A comprehensive study of remodeling of ER is important in understanding M. perniciosa fungus resistance to oxidative stress and its ability to implement a successful infection in T

  13. Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

    PubMed Central

    Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

    2017-01-01

    This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress. PMID:28071725

  14. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis

    PubMed Central

    Hughes, Alexandria; Oxford, Alexandra E.; Tawara, Ken; Jorcyk, Cheryl L.; Oxford, Julia Thom

    2017-01-01

    Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis. PMID:28335520

  15. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.

    PubMed

    Hughes, Alexandria; Oxford, Alexandra E; Tawara, Ken; Jorcyk, Cheryl L; Oxford, Julia Thom

    2017-03-20

    Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.

  16. Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum.

    PubMed

    Kitamura, Takuya; Seki, Naoya; Kihara, Akio

    2017-03-28

    Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

  17. Cloning of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) from Caribbean spiny lobster Panulirus argus

    PubMed Central

    Arunachalam, S. C.; Meleshkevitch, E. A.; Mandal, P. K.; Boudko, D. Y.; Ahearn, G. A.

    2012-01-01

    We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using 45Ca2+ coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca2+-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene. PMID:18825387

  18. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

    PubMed Central

    Zhang, Jintao; Yi, Man; Zha, Longying; Chen, Siqiang; Li, Zhijia; Li, Cheng; Gong, Mingxing; Deng, Hong; Chu, Xinwei; Chen, Jiehua; Zhang, Zheqing; Mao, Limei; Sun, Suxia

    2016-01-01

    Purpose Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. Methods Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. Results Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin

  19. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane

    PubMed Central

    1985-01-01

    We have studied the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in Chinese hamster ovary cells using a cell fractionation assay. We found that transport is dependent on metabolic energy, but that the maintenance of the high differential concentration of cholesterol in the plasma membrane is not an energy-requiring process. We have tested a variety of inhibitors for their effect on cholesterol transport and found that cytochalasin B, colchicine, monensin, cycloheximide, and NH4Cl did not have any effect. The cholesterol transport process shows a sharp temperature dependence; it ceases at 15 degrees C, whereas cholesterol synthesis continues. When synthesis occurs at 15 degrees C, the newly synthesized cholesterol accumulates in the endoplasmic reticulum and in a low density, lipid-rich vesicle fraction. These results suggest that cholesterol is transported via a vesicular system. PMID:4040520

  20. CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response

    PubMed Central

    Caputo, Manuela; Balzerano, Alessio; Arisi, Ivan; D’Onofrio, Mara; Brandi, Rossella; Bongiorni, Silvia; Brancorsini, Stefano; Frontini, Mattia; Proietti-De-Santis, Luca

    2017-01-01

    The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome. PMID:28253359

  1. Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells.

    PubMed

    Yu, Kyeong-Nam; Chang, Seung-Hee; Park, Soo Jin; Lim, Joohyun; Lee, Jinkyu; Yoon, Tae-Jong; Kim, Jun-Sung; Cho, Myung-Haing

    2015-01-01

    Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 μg/mL TiO2-NP for 24 and 48 h. Our results showed that TiO2-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO2-NP promoted toxicity via ER stress. This novel mechanism of TiO2-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application.

  2. GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy

    PubMed Central

    Holczer, Marianna; Bánhegyi, Gábor; Kapuy, Orsolya

    2016-01-01

    The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress. PMID:27992581

  3. Regulation of lipid metabolism via a connection between the endoplasmic reticulum and lipid droplets.

    PubMed

    Suzuki, Michitaka

    2017-01-01

    Lipid droplets (LDs) are ubiquitous organelles that store and supply lipids to regulate cellular lipid homeostasis. Fatty acids are packaged as triglyceride and cholesterol ester into endoplasmic reticulum (ER) membranes to synthesize LDs. Cytosolic LDs move dynamically and interact with organelles, including other LDs. In this process, functional proteins for metabolism are also transferred to LDs. In this review, I focus on interactions between the ER and LDs related to lipid metabolism.

  4. Ultrafast glycerophospholipid-selective transbilayer motion mediated by a protein in the endoplasmic reticulum membrane.

    PubMed

    Buton, X; Morrot, G; Fellmann, P; Seigneuret, M

    1996-03-22

    A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain phospholipids with a new fast translocation assay. Identical results were obtained with both types of probes. Transbilayer motion of glycerophospholipids was found to be much more rapid than previously reported (half-time less than 25 s) and to occur identically for phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Such transport is nonvectorial and leads to a symmetric transbilayer distribution of phospholipids. In contrast, transverse diffusion of sphingomyelin was 1 order of magnitude slower. Phospholipid flip-flop appears to occur by a protein-mediated transport process displaying saturable and competitive behavior. Proteolysis, chemical modification, and competition experiments suggest that this transport process may be related to that previously described in the endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). The relationship between phospholipid flip-flop and nonbilayer structures occurring in the endoplasmic reticulum was also investigated by 31P-NMR. Several conditions were found under which the 31P isotropic NMR signal previously attributed to nonbilayer structures is decreased or abolished, whereas transbilayer diffusion is unaffected, suggesting that the flip-flop process is independent of such structures. It is concluded that flip-flop in the endoplasmic reticulum is mediated by a bidirectional protein transporter with a high efficiency for glycerophospholipids and a low efficiency for sphingomyelin. In vivo, the activity of this transporter would be able to redistribute all changes in phospholipid composition due

  5. Sterol carrier protein-2 localization in endoplasmic reticulum and role in phospholipid formation.

    PubMed

    Starodub, O; Jolly, C A; Atshaves, B P; Roths, J B; Murphy, E J; Kier, A B; Schroeder, F

    2000-10-01

    Although sterol carrier protein-2 (SCP-2; also called nonspecific lipid transfer protein) binds fatty acids and fatty acyl-CoAs, its role in fatty acid metabolism is not fully understood. L-cell fibroblasts stably expressing SCP-2 were used to resolve the relationship between SCP-2 intracellular location and fatty acid transacylation in the endoplasmic reticulum. Indirect immunofluorescence double labeling and laser scanning confocal microscopy detected SCP-2 in peroxisomes > endoplasmic reticulum > mitochondria > lysosomes. SCP-2 enhanced incorporation of exogenous [(3)H]oleic acid into phospholipids and triacylglycerols of overexpressing cells 1.6- and 2.5-fold, respectively, stimulated microsomal incorporation of [1-(14)C]oleoyl-CoA into phosphatidic acid in vitro 13-fold, and exhibited higher specificity for unsaturated versus saturated fatty acyl-CoA. SCP-2 enhanced the rate-limiting step in microsomal phosphatidic acid biosynthesis mediated by glycerol-3-phosphate acyltransferase. SCP-2 also enhanced microsomal acyl-chain remodeling of phosphatidylethanolamine up to fivefold and phosphatidylserine twofold, depending on the specific fatty acyl-CoA, but had no effect on other phospholipid classes. In summary, these results were consistent with a role for SCP-2 in phospholipid synthesis in the endoplasmic reticulum.

  6. Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum.

    PubMed

    Johannes, Ludger; Pezo, Valérie; Mallard, Frédéric; Tenza, Danièle; Wiltz, Aimée; Saint-Pol, Agnès; Helft, Julie; Antony, Claude; Benaroch, Philippe

    2003-05-01

    HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb3, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, beta COP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed.

  7. Endoplasmic reticulum stress: a novel mechanism and therapeutic target for cardiovascular diseases

    PubMed Central

    Liu, Mei-qing; Chen, Zhe; Chen, Lin-xi

    2016-01-01

    Endoplasmic reticulum is a principal organelle responsible for folding, post-translational modifications and transport of secretory, luminal and membrane proteins, thus palys an important rale in maintaining cellular homeostasis. Endoplasmic reticulum stress (ERS) is a condition that is accelerated by accumulation of unfolded/misfolded proteins after endoplasmic reticulum environment disturbance, triggered by a variety of physiological and pathological factors, such as nutrient deprivation, altered glycosylation, calcium depletion, oxidative stress, DNA damage and energy disturbance, etc. ERS may initiate the unfolded protein response (UPR) to restore cellular homeostasis or lead to apoptosis. Numerous studies have clarified the link between ERS and cardiovascular diseases. This review focuses on ERS-associated molecular mechanisms that participate in physiological and pathophysiological processes of heart and blood vessels. In addition, a number of drugs that regulate ERS was introduced, which may be used to treat cardiovascular diseases. This review may open new avenues for studying the pathogenesis of cardiovascular diseases and discovering novel drugs targeting ERS. PMID:26838072

  8. Mammalian vesicle trafficking proteins of the endoplasmic reticulum and Golgi apparatus.

    PubMed

    Hay, J C; Hirling, H; Scheller, R H

    1996-03-08

    Vesicle traffic propagates and maintains distinct subcellular compartments and routes secretory products from their site of synthesis to their final destinations. As a basis for the specificity of vesicular transport reactions, each step in the secretory pathway appears to be handled by a distinct set of evolutionarily conserved proteins. Mammalian proteins responsible for vesicle trafficking at early steps in the secretory pathway are not well understood. In this report, we describe rat sec22 (rsec22) and rat bet1 (rbet1), mammalian sequence homologs of yeast proteins identified as mediators of endoplasmic reticulum-to-Golgi protein transport. rsec22 and rbet1 were expressed widely in mammalian tissues, as anticipated for proteins involved in fundamental membrane trafficking reactions. Recombinant rsec22 and rbet1 proteins behaved as integral membrane components of 28 and 18 kDa, respectively, consistent with their primary structures, which contain a predicted transmembrane domain at or near the carboxyl terminus. Recombinant rsec22 and rbet1 had distinct subcellular localizations, with rsec22 residing on endoplasmic reticulum membranes and rbet1 found on Golgi membranes. Studies with brefeldin A and nocodazole indicated that rbet1 function might involve interaction with or retention in the intermediate compartment. The distinct localizations of rsec22 and rbet1 may reflect their participation in opposite directions of membrane flow between the endoplasmic reticulum and Golgi apparatus.

  9. Valsartan protects HK-2 cells from contrast media-induced apoptosis by inhibiting endoplasmic reticulum stress.

    PubMed

    Peng, Ping-An; Wang, Le; Ma, Qian; Xin, Yi; Zhang, Ou; Han, Hong-Ya; Liu, Xiao-Li; Ji, Qing-Wei; Zhou, Yu-Jie; Zhao, Ying-Xin

    2015-12-01

    Contrast-induced acute kidney injury (CI-AKI) is associated with increasing in-hospital and long-term adverse clinical outcomes in high-risk patients undergoing percutaneous coronary intervention (PCI). Contrast media (CM)-induced renal tubular cell apoptosis is reported to participate in this process by activating endoplasmic reticulum (ER) stress. An angiotensin II type 1 receptor (AT1R) antagonist can alleviate ER stress-induced renal apoptosis in streptozotocin (STZ)-induced diabetic mice and can reduce CM-induced renal apoptosis by reducing oxidative stress and reversing the enhancement of bax mRNA and the reduction of bcl-2 mRNA, but the effect of the AT1R blocker on ER stress in the pathogenesis of CI-AKI is still unknown. In this study, we explored the effect of valsartan on meglumine diatrizoate-induced human renal tubular cell apoptosis by measuring changes in ER stress-related biomarkers. The results showed that meglumine diatrizoate caused significant cell apoptosis by up-regulating the expression of ER stress markers, including glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) and caspase 12, in a time- and dose-dependent manner, which could be alleviated by preincubation with valsartan. In conclusion, valsartan had a potential nephroprotective effect on meglumine diatrizoate-induced renal cell apoptosis by inhibiting ER stress.

  10. Algae Undaria pinnatifida Protects Hypothalamic Neurons against Endoplasmic Reticulum Stress through Akt/mTOR Signaling.

    PubMed

    Kim, Jongwan; Moon, Il Soo; Goo, Tae-Won; Moon, Seong-Su; Seo, Minchul

    2015-11-25

    Increased endoplasmic reticulum (ER) stress is known to be one of the causes of hypothalamic neuronal damage, as well as a cause of metabolic disorders such as obesity and diabetes. Recent evidence has suggested that Undaria pinnatifida (UP), an edible brown algae, has antioxidant activity. However, the neuroprotective effect of UP has yet to be examined. In this study, to investigate the neuroprotective effect of UP on ER stress-induced neuronal damage in mouse hypothalamic neurons, mice immortal hypothalamic neurons (GT1-7) were incubated with extract of UP. ER stress was induced by treating with tunicamycin. Tunicamycin induced apoptotic cell death was compared with the vehicle treatment through excessive ER stress. However UP protected GT1-7 cells from cell death, occurring after treatment with tunicamycin by reducing ER stress. Treatment with UP resulted in reduced increment of ATF6 and CHOP, and recovered the decrease of phosphorylation of Akt/mTOR by tunicamycin and the increment of autophagy. These results show that UP protects GT1-7 cells from ER stress induced cell death through the Akt/mTOR pathway. The current study suggests that UP may have a beneficial effect on cerebral neuronal degeneration in metabolic diseases with elevated ER stress.

  11. PPARδ Is Required for Exercise to Attenuate Endoplasmic Reticulum Stress and Endothelial Dysfunction in Diabetic Mice.

    PubMed

    Cheang, Wai San; Wong, Wing Tak; Zhao, Lei; Xu, Jian; Wang, Li; Lau, Chi Wai; Chen, Zhen Yu; Ma, Ronald Ching Wan; Xu, Aimin; Wang, Nanping; Tian, Xiao Yu; Huang, Yu

    2017-02-01

    Physical activity has profound benefits on health, especially on cardiometabolic wellness. Experiments in rodents with trained exercise have shown that exercise improves vascular function and reduces vascular inflammation by modulating the balance between nitric oxide (NO) and oxidative stress. However, the upstream regulator of exercise-induced vascular benefits is unclear. We aimed to investigate the involvement of peroxisome proliferator-activated receptor δ (PPARδ) in exercise-induced vascular functional improvement. We show that PPARδ is a crucial mediator for exercise to exert a beneficial effect on the vascular endothelium in diabetic mice. In db/db mice and high-fat diet-induced obese mice, 4 weeks of treadmill exercise restored endothelium-dependent vasodilation of aortas and flow-mediated vasodilation in mesenteric resistance arteries, whereas genetic ablation of Ppard abolished such improvements. Exercise induces AMPK activation and subsequent PPARδ activation, which help to reduce endoplasmic reticulum (ER) and oxidative stress, thus increasing NO bioavailability in endothelial cells and vascular tissues. Chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid decrease ER stress and protect against endothelial dysfunction in diabetic mice. The results demonstrate that PPARδ-mediated inhibition of ER stress contributes to the vascular benefits of exercise and provides potentially effective targets for treating diabetic vasculopathy.

  12. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayoshi; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2013-02-01

    Highlights: ► Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ► Adipose lipin-1 expression is reduced in obesity. ► ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ► Activation of PPAR-γ recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  13. Inhibition of endoplasmic reticulum stress improves coronary artery function in the spontaneously hypertensive rats

    PubMed Central

    Choi, Soo-Kyoung; Lim, Mihwa; Byeon, Seon-Hee; Lee, Young-Ho

    2016-01-01

    Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of cardiovascular complications. However, the role and mechanisms of ER stress in hypertension remain unclear. Thus, we hypothesized that enhanced ER stress contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). Sixteen-week old male SHRs and Wistar Kyoto Rats (WKYs) were used in this study. The SHRs were treated with ER stress inhibitor (Tauroursodeoxycholic acid; TUDCA, 100 mg/kg/day) for two weeks. There was a decrease in systolic blood pressure in SHR treated with TUDCA. The pressure-induced myogenic tone was significantly increased, whereas endothelium-dependent relaxation was significantly attenuated in SHR compared with WHY. Interestingly, treatment of ER stress inhibitor normalized myogenic responses and endothelium-dependent relaxation in SHR. These data were associated with an increase in expression or phosphorylation of ER stress markers (Bip, ATF6, CHOP, IRE1, XBP1, PERK, and eIF2α) in SHRs, which were reduced by TUDCA treatment. Furthermore, phosphorylation of MLC20 was increased in SHRs, which was reduced by the treatment of TUDCA. Therefore, our results suggest that ER stress could be a potential target for hypertension. PMID:27550383

  14. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  15. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    SciTech Connect

    Pan, Mu-Yun; Shen, Yuh-Chiang; Lu, Chien-Hsing; Yang, Shu-Yi; Ho, Tsing-Fen; Peng, Yu-Ta; Chang, Chia-Che

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified as an

  16. Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis.

    PubMed

    Lumley, Emily C; Osborn, Acadia R; Scott, Jessica E; Scholl, Amanda G; Mercado, Vicki; McMahan, Young T; Coffman, Zachary G; Brewster, Jay L

    2017-01-01

    The endoplasmic reticulum (ER) has the ability to signal organelle dysfunction via a complex signaling network known as the unfolded protein response (UPR). In this work, hamster fibroblast cells exhibiting moderate levels of ER stress were compared to those exhibiting severe ER stress. Inhibition of N-linked glycosylation was accomplished via a temperature-sensitive mutation in the Dad1 subunit of the oligosaccharyltransferase (OST) complex or by direct inhibition with tunicamycin (Tm). Temperature shift (TS) treatment generated weak activation of ER stress signaling when compared to doses of Tm that are typically used in ER stress studies (500-1000 nM). A dose-response analysis of key ER stress signaling mediators, inositol-requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), revealed 20-40 nM of Tm to generate activation intensity similar to TS treatment. In parental BHK21 cells, moderate (20-40 nM) and high doses (200-1000 nM) of Tm were compared to identify physiological and signaling-based differences in stress response. Inhibition of ER Ca(2+) release via ITPR activity with 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) was sufficient to protect against apoptosis induced by moderate but not higher doses of Tm. Analysis of kinase activation over a range of Tm exposures revealed the p38 stress-activated protein kinase (SAPK) to display increasing activation with Tm dosage. Interestingly, Tm induced the extracellular regulated kinases (Erk1/2) only at moderate doses of Tm. Inhibition of ER transmembrane stress sensors (IRE1, PERK) or cytosolic signaling mediators (p38, Jnk1, Erk1/2) was used to evaluate pathways involved in apoptosis activation during ER stress. Inhibition of either PERK or p38 was sufficient to reduce cell death and apoptosis induced by moderate, but not high, doses of Tm. During ER stress, cells exhibited a rapid decline in anti-apoptotic Mcl-1 and survivin proteins. Inhibition of

  17. Hypothyroidism minimizes the effects of acute hepatic failure caused by endoplasmic reticulum stress and redox environment alterations in rats.

    PubMed

    Blas-Valdivia, Vanessa; Cano-Europa, Edgar; Martinez-Perez, Yoalli; Lezama-Palacios, Ruth; Franco-Colin, Margarita; Ortiz-Butron, Rocio

    2015-10-01

    The aim of this study was to investigate if a protective effect from hypothyroidism in acute liver failure resulted from reduced endoplasmic reticulum stress and changes to the redox environment. Twenty male Sprague-Dawley rats were divided in four groups: (1) euthyroid (sham surgery), (2) hypothyroid, (3) euthyroid (sham surgery)+thioacetamide and (4) hypothyroid+thioacetamide. Hypothyroidism was confirmed two weeks after thyroidectomy, and thioacetamide (TAA) (400mg/kg, ip) was administrated to the appropriate groups for three days with supportive therapy. Grades of encephalopathy in all animals were determined using behavioral tests. Animals were decapitated and their blood was obtained to assess liver function. The liver was dissected: the left lobe was used for histology and the right lobe was frozen for biochemical assays. Body weight, rectal temperature and T4 concentration were lower in hypothyroid groups. When measurements of oxidative stress markers, redox environment, γ-glutamylcysteine synthetase and glutathione-S-transferase were determined, we observed that hypothyroid animals with TAA compensated better with oxidative damage than euthyroid animals treated with TAA. Furthermore, we measured reduced expressions of GADD34, caspase-12 and GRP78 and subsequently less hypothyroidism-induced cellular damage in hypothyroid animals. We conclude that hypothyroidism protects against hepatic damage caused by TAA because it reduces endoplasmic reticulum stress and changes to the redox environment.

  18. Regulation and Quality Control of Adiponectin Assembly by Endoplasmic Reticulum Chaperone ERp44*

    PubMed Central

    Hampe, Lutz; Radjainia, Mazdak; Xu, Cheng; Harris, Paul W. R.; Bashiri, Ghader; Goldstone, David C.; Brimble, Margaret A.; Wang, Yu; Mitra, Alok K.

    2015-01-01

    Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys39 located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12–18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys39 and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control. PMID:26060250

  19. Alteration of endoplasmic reticulum lipid rafts contributes to lipotoxicity in pancreatic β-cells.

    PubMed

    Boslem, Ebru; Weir, Jacquelyn M; MacIntosh, Gemma; Sue, Nancy; Cantley, James; Meikle, Peter J; Biden, Trevor J

    2013-09-13

    Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.

  20. Diet-induced obesity induces endoplasmic reticulum stress and insulin resistance in the amygdala of rats☆

    PubMed Central

    Castro, Gisele; C. Areias, Maria Fernanda; Weissmann, Lais; Quaresma, Paula G.F.; Katashima, Carlos K.; Saad, Mario J.A.; Prada, Patricia O.

    2013-01-01

    Insulin acts in the hypothalamus, decreasing food intake (FI) by the IR/PI3K/Akt pathway. This pathway is impaired in obese animals and endoplasmic reticulum (ER) stress and low-grade inflammation are possible mechanisms involved in this impairment. Here, we highlighted the amygdala as an important brain region for FI regulation in response to insulin. This regulation was dependent on PI3K/AKT pathway similar to the hypothalamus. Insulin was able to decrease neuropeptide Y (NPY) and increase oxytocin mRNA levels in the amygdala via PI3K, which may contribute to hypophagia. Additionally, obese rats did not reduce FI in response to insulin and AKT phosphorylation was decreased in the amygdala, suggesting insulin resistance. Insulin resistance was associated with ER stress and low-grade inflammation in this brain region. The inhibition of ER stress with PBA reverses insulin action/signaling, decreases NPY and increases oxytocin mRNA levels in the amygdala from obese rats, suggesting that ER stress is probably one of the mechanisms that induce insulin resistance in the amygdala. PMID:24251109

  1. Identification of a Novel Endoplasmic Reticulum Stress Response Element Regulated by XBP1*

    PubMed Central

    Misiewicz, Michael; Déry, Marc-André; Foveau, Bénédicte; Jodoin, Julie; Ruths, Derek; LeBlanc, Andréa C.

    2013-01-01

    Understanding the regulatory mechanisms mediating PRNP gene expression is highly relevant to elucidating normal cellular prion protein (PrP) function(s) and the transmissibility of prion protein neurodegenerative diseases. Here, luciferase reporter assays showed that an endoplasmic reticulum stress element (ERSE)-like element, CCAAT-N26-CCACG in the human PRNP promoter, is regulated by ER stress and X-box-binding protein 1 (XBP1) but not by activating transcription factor 6 α (ATF6α). Bioinformatics identified the ERSE-26 motif in 37 other human genes in the absence of canonical ERSE sites except for three genes. Several of these genes are associated with a synaptic function or are involved in oxidative stress. Brefeldin A, tunicamycin, and thapsigargin ER stressors induced gene expression of PRNP and four randomly chosen ERSE-26-containing genes, ERLEC1, GADD45B, SESN2, and SLC38A5, in primary human neuron cultures or in the breast carcinoma MCF-7 cell line, although the level of the response depends on the gene analyzed, the genetic background of the cells, the cell type, and the ER stressor. Overexpression of XBP1 increased, whereas siRNA knockdown of XBP1 considerably reduced, PRNP and ERLEC1 mRNA levels in MCF-7 cells. Taken together, these results identify a novel ER stress regulator, which implicates the ER stress response in previously unrecognized cellular functions. PMID:23737521

  2. Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells

    PubMed Central

    Montagnani Marelli, Marina; Marzagalli, Monica; Moretti, Roberta M.; Beretta, Giangiacomo; Casati, Lavinia; Comitato, Raffaella; Gravina, Giovanni L.; Festuccia, Claudio; Limonta, Patrizia

    2016-01-01

    Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma. PMID:27461002

  3. The reticulons: Guardians of the structure and function of the endoplasmic reticulum

    SciTech Connect

    Di Sano, Federica; Bernardoni, Paolo; Piacentini, Mauro

    2012-07-01

    The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by a specific class of curvature stabilizing proteins, the reticulons and DP1; however it is still unclear how the sheets are assembled. The ER is the cellular compartment responsible for secretory and membrane protein synthesis. The reducing conditions of ER lead to the intra/inter-chain formation of new disulphide bonds into polypeptides during protein folding assessed by enzymatic or spontaneous reactions. Moreover, ER represents the main intracellular calcium storage site and it plays an important role in calcium signaling that impacts many cellular processes. Accordingly, the maintenance of ER function represents an essential condition for the cell, and ER morphology constitutes an important prerogative of it. Furthermore, it is well known that ER undergoes prominent shape transitions during events such as cell division and differentiation. Thus, maintaining the correct ER structure is an essential feature for cellular physiology. Now, it is known that proper ER-associated proteins play a fundamental role in ER tubules formation. Among these ER-shaping proteins are the reticulons (RTN), which are acquiring a relevant position. In fact, beyond the structural role of reticulons, in very recent years new and deeper functional implications of these proteins are emerging in relation to their involvement in several cellular processes.

  4. ROS mediated crosstalk between endoplasmic reticulum and mitochondria by Phloxine B under environmental UV irradiation.

    PubMed

    Goyal, Shruti; Amar, Saroj Kumar; Srivastav, Ajeet Kumar; Chopra, Deepti; Pal, Manish Kumar; Arjaria, Nidhi; Ray, Ratan Singh

    2016-08-01

    Phloxine B (PhB) is a most commonly used dye in cosmetic products throughout the world. It shows an absorption in visible and ultraviolet radiations. PhB was photodegraded within 4h of UV exposure. It generates reactive oxygen species (ROS) photochemically and intracellularly. Photosensitized PhB caused dose dependent cell viability reduction of human keratinocyte cell line (HaCaT) which was measured through MTT (75.4%) and NRU (77.3%) assays. It also induces cell cycle arrest and DNA damage. Photosensitized PhB induces Ca(2+) release from endoplasmic reticulum (ER). It causes the upregulation of ER stress marker genes ATF6 (1.79 fold) and CHOP (1.93 fold) at transcription levels. The similar response of ATF6 (3.6 fold) and CHOP (2.38 fold) proteins was recorded at translation levels. CHOP targeted the mitochondria and reduced the mitochondrial membrane potential analyzed through JC-1 staining. It further increases Bax/Bcl2 ratio (3.58 fold) and promotes the release of cytochrome c, finally leads to caspase-dependent apoptosis. Upregulation of APAF1 (1.79 fold) in PhB treated cells under UV B exposure supports the mitochondrial-mediated apoptotic cell death. The results support the involvement of ER and mitochondria in ROS mediated PhB phototoxicity. Therefore, the use of PhB in cosmetic products may be deleterious to users during sunlight exposure.

  5. Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response.

    PubMed

    Yi, Hong-Wei; Ma, Yu-Xiang; Wang, Xiao-Ning; Wang, Cui-Fen; Lu, Jian; Cao, Wei; Wu, Xu-Dong

    2015-04-01

    Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP(-/-) mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.

  6. Endoplasmic reticulum stress protects human thyroid carcinoma cell lines against ionizing radiation-induced apoptosis.

    PubMed

    Wu, Xin-Yu; Fan, Rui-Tai; Yan, Xin-Hui; Cui, Jing; Xu, Jun-Ling; Gu, Hao; Gao, Yong-Ju

    2015-03-01

    Radiotherapy is one of the most effective forms of cancer treatment, used in the treatment of a number of malignant tumors. However, the resistance of tumor cells to ionizing radiation remains a major therapeutic problem and the critical mechanisms determining radiation resistance are poorly defined. In the present study, a cellular endoplasmic reticulum (ER) stress microenvironment was established through the pretreatment of cultured thyroid cancer cells with tunicamycin (TM) and thapsigargin (TG), in order to mimic the ER stress response in a tumor microenvironment. This microenviroment was confirmed through the X‑box binding protein 1 splice process, glucose‑regulated protein 78 kD and ER degradation‑enhancing α‑mannosidase‑like mRNA expression. A clonogenic assay was used to measure cancer cell resistance to 60Co‑γ following TM pretreatment; in addition, human C/EBP homologous protein (CHOP) mRNA expression was determined and apoptosis assays were performed. The results showed that TM or TG pretreatment inhibited CHOP expression and reduced the apoptotic rate of cells. Furthermore, the results demonstrated that the induced ER stress response rendered cancer cells more resistant to ionizing radiation‑induced apoptosis. Therefore, the ER stress pathway may be a potential therapeutic target in order to improve the clinical efficiency of radiotherapy.

  7. Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

    SciTech Connect

    Tang, C.-H.; Chiu, Y.-C.; Huang, C.-F.; Chen, Y.-W.; Chen, P.-C.

    2009-12-01

    Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

  8. Androgen Mediated Regulation of Endoplasmic Reticulum-Associated Degradation and its Effects on Prostate Cancer

    PubMed Central

    Erzurumlu, Yalcin; Ballar, Petek

    2017-01-01

    The endoplasmic reticulum (ER) comprises thirty percent of the newly translated proteins in eukaryotic cells. The quality control mechanism within the ER distinguishes between properly and improperly folded proteins and ensures that unwanted proteins are retained in the ER and subsequently degraded through ER-associated degradation (ERAD). Besides cleaning of misfolded proteins ERAD is also important for physiological processes by regulating the abundance of normal proteins of the ER. Thus it is important to unreveal the regulation patterns of ERAD. Here, we describe that ERAD pathway is regulated by androgen, where its inhibitor SVIP was downregulated, all other ERAD genes were upregulated. Consistently, androgen treatment increased the degradation rate of ERAD substrates. Using several independent techniques, we showed that this regulation is through androgen receptor transactivation. ERAD genes found to be upregulated in prostate cancer tissues and silencing expression of Hrd1, SVIP, and gp78 reduced the in vitro migration and malignant transformation of LNCaP cells. Our data suggests that expression levels of ERAD components are regulated by androgens, that promotes ERAD proteolytic activity, which is positively related with prostate tumorigenesis. PMID:28091582

  9. Junctate boosts phagocytosis by recruiting endoplasmic reticulum Ca2+ stores near phagosomes.

    PubMed

    Guido, Daniele; Demaurex, Nicolas; Nunes, Paula

    2015-11-15

    Local intracellular Ca(2+) elevations increase the efficiency of phagocytosis, a process that is essential for innate and adaptive immunity. These local Ca(2+) elevations are generated in part by the store-operated Ca(2+) entry (SOCE) sensor STIM1, which recruits endoplasmic reticulum (ER) cisternae to phagosomes and opens phagosomal Ca(2+) channels at ER-phagosome junctions. However, residual ER-phagosome contacts and periphagosomal Ca(2+) hotspots remain in Stim1(-/-) cells. Here, we tested whether junctate (also called ASPH isoform 8), a molecule that targets STIM1 to ER-plasma-membrane contacts upon Ca(2+)-store depletion, cooperates with STIM1 at phagosome junctions. Junctate expression in Stim1(-/-) and Stim1(-/-); Stim2(-/-) phagocytic fibroblasts increased phagocytosis and periphagosomal Ca(2+) elevations, yet with only a minimal impact on global SOCE. These Ca(2+) hotspots were only marginally reduced by the SOCE channel blocker lanthanum chloride (La(3+)) but were abrogated by inositol trisphosphate receptor inhibitors 2-APB and xestospongin-C, revealing that unlike STIM1-mediated hotspots, junctate-mediated Ca(2+) originates predominantly from periphagosomal Ca(2+) stores. Accordingly, junctate accumulates near phagosomes and elongates ER-phagosome junctions in Stim1(-/-) cells. Thus, junctate mediates an alternative mechanism for generating localized Ca(2+) elevations within cells, promoting Ca(2+) release from internal stores recruited to phagosomes, thereby boosting phagocytosis.

  10. Cellular Pathology of Pelizaeus-Merzbacher Disease Involving Chaperones Associated with Endoplasmic Reticulum Stress

    PubMed Central

    Inoue, Ken

    2017-01-01

    Disease-causing mutations in genes encoding membrane proteins may lead to the production of aberrant polypeptides that accumulate in the endoplasmic reticulum (ER). These mutant proteins have detrimental conformational changes or misfolding events, which result in the triggering of the unfolded protein response (UPR). UPR is a cellular pathway that reduces ER stress by generally inhibiting translation, increasing ER chaperones levels, or inducing cell apoptosis in severe ER stress. This process has been implicated in the cellular pathology of many neurological disorders, including Pelizaeus-Merzbacher disease (PMD). PMD is a rare pediatric disorder characterized by the failure in the myelination process of the central nervous system (CNS). PMD is caused by mutations in the PLP1 gene, which encodes a major myelin membrane protein. Severe clinical PMD phenotypes appear to be the result of cell toxicity, due to the accumulation of PLP1 mutant proteins and not due to the lack of functional PLP1. Therefore, it is important to clarify the pathological mechanisms by which the PLP1 mutants negatively impact the myelin-generating cells, called oligodendrocytes, to overcome this devastating disease. This review discusses how PLP1 mutant proteins change protein homeostasis in the ER of oligodendrocytes, especially focusing on the reaction of ER chaperones against the accumulation of PLP1 mutant proteins that cause PMD. PMID:28286750

  11. Mapping the Interactome of a Major Mammalian Endoplasmic Reticulum Heat Shock Protein 90

    PubMed Central

    Hong, Feng; Mohammad Rachidi, Saleh; Lundgren, Debbie; Han, David; Huang, Xiu; Zhao, Hongyu; Kimura, Yayoi; Hirano, Hisashi; Ohara, Osamu; Udono, Heichiiro; Meng, Songdong; Liu, Bei; Li, Zihai

    2017-01-01

    Up to 10% of cytosolic proteins are dependent on the mammalian heat shock protein 90 (HSP90) for folding. However, the interactors of its endoplasmic reticulum (ER) paralogue (gp96, Grp94 and HSP90b1) has not been systematically identified. By combining genetic and biochemical approaches, we have comprehensively mapped the interactome of gp96 in macrophages and B cells. A total of 511 proteins were reduced in gp96 knockdown cells, compared to levels observed in wild type cells. By immunoprecipitation, we found that 201 proteins associated with gp96. Gene Ontology analysis indicated that these proteins are involved in metabolism, transport, translation, protein folding, development, localization, response to stress and cellular component biogenesis. While known gp96 clients such as integrins, Toll-like receptors (TLRs) and Wnt co-receptor LRP6, were confirmed, cell surface HSP receptor CD91, TLR4 pathway protein CD180, WDR1, GANAB and CAPZB were identified as potentially novel substrates of gp96. Taken together, our study establishes gp96 as a critical chaperone to integrate innate immunity, Wnt signaling and organ development. PMID:28056051

  12. Endoplasmic Reticulum Stress and the Unfolded Protein Responses in Retinal Degeneration

    PubMed Central

    Zhang, Sarah X.; Sanders, Emily; Fliesler, Steven J.; Wang, Joshua J.

    2014-01-01

    The endoplasmic reticulum (ER) is the primary intracellular organelle responsible for protein and lipid biosynthesis, protein folding and trafficking, calcium homeostasis, and several other vital processes in cell physiology. Disturbance in ER function results in ER stress and subsequent activation of the unfolded protein response (UPR). The UPR up-regulates ER chaperones, reduces protein translation, and promotes clearance of cytotoxic misfolded proteins to restore ER homeostasis. If this vital process fails, the cell will be signaled to enter apoptosis, resulting in cell death. Sustained ER stress also can trigger an inflammatory response and exacerbate oxidative stress, both of which contribute synergistically to tissue damage. Studies performed over the past decade have implicated ER stress in a broad range of human diseases, including neurodegenerative diseases, cancer, diabetes, and vascular disorders. Several of these diseases also entail retinal dysfunction and degeneration caused by injury to retinal neurons and/or to the blood vessels that supply retinal cells with nutrients, trophic and homeostatic factors, oxygen, and other essential molecules, as well as serving as a conduit for removal of waste products and potentially toxic substances from the retina. Collectively, such injuries represent the leading cause of blindness world-wide in all age groups. Herein, we summarize recent progress on the study of ER stress and UPR signaling in retinal biology and discuss the molecular mechanisms and the potential clinical applications of targeting ER stress as a new therapeutic approach to prevent and treat neuronal degeneration in the retina. PMID:24792589

  13. Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

    PubMed Central

    De Miguel, Carmen; Hamrick, William C.; Hobbs, Janet L.; Pollock, David M.; Carmines, Pamela K.; Pollock, Jennifer S.

    2017-01-01

    Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury. PMID:28230089

  14. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum

    PubMed Central

    Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A.

    2011-01-01

    The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly. PMID:21357747

  15. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    PubMed Central

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal MD; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-01-01

    Impaired homeostasis of lysosomal Ca2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca2+ are not known. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome. DOI: http://dx.doi.org/10.7554/eLife.15887.001 PMID:27213518

  16. Distinct mechanisms controlling rough and smooth endoplasmic reticulum contacts with mitochondria.

    PubMed

    Wang, Peter T C; Garcin, Pierre O; Fu, Min; Masoudi, Matthew; St-Pierre, Pascal; Panté, Nelly; Nabi, Ivan R

    2015-08-01

    Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.

  17. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    SciTech Connect

    Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin

    2013-10-15

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  18. Endoplasmic reticulum stress-regulated CXCR3 pathway mediates inflammation and neuronal injury in acute glaucoma.

    PubMed

    Ha, Y; Liu, H; Xu, Z; Yokota, H; Narayanan, S P; Lemtalsi, T; Smith, S B; Caldwell, R W; Caldwell, R B; Zhang, W

    2015-10-08

    Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1β and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia.

  19. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress.

    PubMed

    Mecha, M; Torrao, A S; Mestre, L; Carrillo-Salinas, F J; Mechoulam, R; Guaza, C

    2012-06-28

    Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation.

  20. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

    PubMed Central

    Mecha, M; Torrao, A S; Mestre, L; Carrillo-Salinas, F J; Mechoulam, R; Guaza, C

    2012-01-01

    Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the ‘oligoprotective' effects of CBD during inflammation. PMID:22739983

  1. Reactive Oxygen Species, Endoplasmic Reticulum Stress and Mitochondrial Dysfunction: The Link with Cardiac Arrhythmogenesis

    PubMed Central

    Tse, Gary; Yan, Bryan P.; Chan, Yin W. F.; Tian, Xiao Yu; Huang, Yu

    2016-01-01

    Background: Cardiac arrhythmias represent a significant problem globally, leading to cerebrovascular accidents, myocardial infarction, and sudden cardiac death. There is increasing evidence to suggest that increased oxidative stress from reactive oxygen species (ROS), which is elevated in conditions such as diabetes and hypertension, can lead to arrhythmogenesis. Method: A literature review was undertaken to screen for articles that investigated the effects of ROS on cardiac ion channel function, remodeling and arrhythmogenesis. Results: Prolonged endoplasmic reticulum stress is observed in heart failure, leading to increased production of ROS. Mitochondrial ROS, which is elevated in diabetes and hypertension, can stimulate its own production in a positive feedback loop, termed ROS-induced ROS release. Together with activation of mitochondrial inner membrane anion channels, it leads to mitochondrial depolarization. Abnormal function of these organelles can then activate downstream signaling pathways, ultimately culminating in altered function or expression of cardiac ion channels responsible for generating the cardiac action potential (AP). Vascular and cardiac endothelial cells become dysfunctional, leading to altered paracrine signaling to influence the electrophysiology of adjacent cardiomyocytes. All of these changes can in turn produce abnormalities in AP repolarization or conduction, thereby increasing likelihood of triggered activity and reentry. Conclusion: ROS plays a significant role in producing arrhythmic substrate. Therapeutic strategies targeting upstream events include production of a strong reducing environment or the use of pharmacological agents that target organelle-specific proteins and ion channels. These may relieve oxidative stress and in turn prevent arrhythmic complications in patients with diabetes, hypertension, and heart failure. PMID:27536244

  2. Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

    PubMed

    Chen, Fenglei; Li, Qian; Zhang, Zhe; Lin, Pengfei; Lei, Lanjie; Wang, Aihua; Jin, Yaping

    2015-08-20

    Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

  3. Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells.

    PubMed

    Montagnani Marelli, Marina; Marzagalli, Monica; Moretti, Roberta M; Beretta, Giangiacomo; Casati, Lavinia; Comitato, Raffaella; Gravina, Giovanni L; Festuccia, Claudio; Limonta, Patrizia

    2016-07-27

    Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma.

  4. Proline biosynthesis is required for endoplasmic reticulum stress tolerance in Saccharomyces cerevisiae.

    PubMed

    Liang, Xinwen; Dickman, Martin B; Becker, Donald F

    2014-10-03

    The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP(+)/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.

  5. Aberrant Neuronal Differentiation and Inhibition of Dendrite Outgrowth Resulting from Endoplasmic Reticulum Stress

    PubMed Central

    Kawada, Koichi; Iekumo, Takaaki; Saito, Ryo; Kaneko, Masayuki; Mimori, Seisuke; Nomura, Yasuyuki; Okuma, Yasunobu

    2014-01-01

    Neural stem cells (NSCs) play an essential role in development of the central nervous system. Endoplasmic reticulum (ER) stress induces neuronal death. After neuronal death, neurogenesis is generally enhanced to repair the damaged regions. However, it is unclear whether ER stress directly affects neurogenesis-related processes such as neuronal differentiation and dendrite outgrowth. We evaluated whether neuronal differentiation and dendrite outgrowth were regulated by HRD1, a ubiquitin ligase that was induced under mild conditions of tunicamycin-induced ER stress. Neurons were differentiated from mouse embryonic carcinoma P19 cells by using retinoic acid. The differentiated cells were cultured for 8 days with or without tunicamycin and HRD1 knockdown. The ER stressor led to markedly increased levels of ER stress. ER stress increased the expression levels of neuronal marker βIII-tubulin in 8-day-differentiated cells. However, the neurites of dendrite marker microtubule-associated protein-2 (MAP-2)-positive cells appeared to retract in response to ER stress. Moreover, ER stress markedly reduced the dendrite length and MAP-2 expression levels, whereas it did not affect the number of surviving mature neurons. In contrast, HRD1 knockdown abolished the changes in expression of proteins such as βIII-tubulin and MAP-2. These results suggested that ER stress caused aberrant neuronal differentiation from NSCs followed by the inhibition of neurite outgrowth. These events may be mediated by increased HRD1 expression. PMID:24723324

  6. Endoplasmic Reticulum Quality Control and Systemic Amyloid Disease: Impacting Protein Stability from the Inside Out

    PubMed Central

    Chen, John J.; Genereux, Joseph C.; Wiseman, R. Luke

    2015-01-01

    The endoplasmic reticulum (ER) is responsible for regulating proteome integrity throughout the secretory pathway. The ER protects downstream secretory environments such as the extracellular space by partitioning proteins between ER protein folding, trafficking and degradation pathways in a process called ER quality control. In this process, ER quality control factors identify misfolded, aggregation-prone protein conformations and direct them towards ER protein folding or degradation, reducing their secretion to the extracellular space where they could further misfold or aggregate into proteotoxic conformations. Despite the general efficiency of ER quality control, many human diseases, such as the systemic amyloidoses, involve aggregation of destabilized, aggregation-prone proteins in the extracellular space. A common feature for all systemic amyloid diseases is the ability for amyloidogenic proteins to evade ER quality control and be efficiently secreted. The efficient secretion of these amyloidogenic proteins increases their serum concentrations available for the distal proteotoxic aggregation characteristic of these diseases. This indicates that ER quality control, and the regulation thereof, is a critical determinant in defining the onset and pathology of systemic amyloid diseases. Here, we discuss the pathologic and potential therapeutic relationship between ER quality control, protein secretion and distal deposition of amyloidogenic proteins involved in systemic amyloid diseases. Furthermore, we present evidence that the Unfolded Protein Response, the stress-responsive signaling pathway that regulates ER quality control, is involved in the pathogenesis of systemic amyloid diseases and represents a promising emerging therapeutic target to intervene in this class of human disease. PMID:26018985

  7. Efavirenz Causes Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Endothelial Cells.

    PubMed

    Weiß, Marlene; Kost, Bernd; Renner-Müller, Ingrid; Wolf, Eckhard; Mylonas, Ioannis; Brüning, Ansgar

    2016-01-01

    The non-nucleoside reverse transcriptase inhibitor efavirenz is a widely prescribed antiretroviral drug used in combined antiretroviral therapy. Despite being an essential and life-saving medication, the required lifelong use of HIV drugs has been associated with a variety of adverse effects, including disturbances in lipid metabolism and increased cardiovascular risk. Efavirenz belongs to those HIV drugs for which cardiovascular and endothelial dysfunctions have been reported. It is here shown that elevated concentrations of efavirenz can inhibit endothelial meshwork formation on extracellular matrix gels by normal and immortalized human umbilical vein cells. This inhibition was associated with an increase in oxidative stress markers, endoplasmic reticulum (ER) stress markers, and autophagy. Induction of ER stress occurred at pharmacologically relevant concentrations of efavirenz and resulted in reduced proliferation and cell viability of endothelial cells, which worsened in the presence of elevated efavirenz concentrations. In combination with the HIV protease inhibitor nelfinavir, both oxidative stress and ER stress became elevated in endothelial cells. These data indicate that pharmacologically relevant concentrations of efavirenz can impair cell viability of endothelial cells and that these effects may be aggravated by either elevated concentrations of efavirenz or by a combined use of efavirenz with other oxidative stress-inducing medications.

  8. Bap31 enhances the endoplasmic reticulum export and quality control of human class I MHC molecules.

    PubMed

    Ladasky, John J; Boyle, Sarah; Seth, Malini; Li, Hewang; Pentcheva, Tsvetelina; Abe, Fumiyoshi; Steinberg, Steven J; Edidin, Michael

    2006-11-01

    The assembly of class I MHC molecules and their export from the endoplasmic reticulum (ER) is governed by chaperones and accessory proteins. We present evidence that the putative cargo receptor protein Bap31 participates in the transport and the quality control of human class I molecules. Transfection of the human adenocarcinoma cell line HeLa with yellow fluorescent protein-Bap31 chimeras increased surface levels of class I in a dose-dependent manner, by as much as 3.7-fold. The increase in surface class I resulted from an increase in the rate of export of newly synthesized class I molecules to the cell surface and from an increase in the stability of the exported molecules. We propose that Bap31 performs quality control on class I molecules in two distinct phases: first, by exporting peptide-loaded class I molecules to the ER/Golgi intermediate compartment, and second, by retrieving class I molecules that have lost peptides in the acidic post-ER environment. This function of Bap31 is conditional or redundant, because we find that Bap31 deficiency does not reduce surface class I levels. Overexpression of the Bap31 homolog, Bap29, decreases surface class levels in HeLa, indicating that it does not substitute for Bap31.

  9. High fat diet dysregulates microRNA-17-5p and triggers retinal inflammation: Role of endoplasmic-reticulum-stress

    PubMed Central

    Coucha, Maha; Mohamed, Islam N; Elshaer, Sally L; Mbata, Osinakachuk; Bartasis, Megan L; El-Remessy, Azza B

    2017-01-01

    AIM To elucidate how high diet-induced endoplasmic reticulum-stress upregulates thioredoxin interacting protein expression in Müller cells leading to retinal inflammation. METHODS Male C57Bl/J mice were fed either normal diet or 60% high fat diet for 4-8 wk. During the 4 wk study, mice received phenyl-butyric acid (PBA); endoplasmic reticulum-stress inhibitor; for 2 wk. Insulin resistance was assessed by oral glucose tolerance. Effects of palmitate-bovine serum albumin (BSA) (400 μmol/L) were examined in retinal Müller glial cell line and primary Müller cells isolated from wild type and thioredoxin interacting protein knock-out mice. Expression of thioredoxin interacting protein, endoplasmic reticulum-stress markers, miR-17-5p mRNA, as well as nucleotide-binding oligomerization domain-like receptor protein (NLRP3) and IL1β protein was determined. RESULTS High fat diet for 8 wk induced obesity and insulin resistance evident by increases in body weight and impaired glucose tolerance. By performing quantitative real-time polymerase chain reaction, we found that high fat diet triggered the expression of retinal endoplasmic reticulum-stress markers (P < 0.05). These effects were associated with increased thioredoxin interacting protein and decreased miR-17-5p expression, which were restored by inhibiting endoplasmic reticulum-stress with PBA (P < 0.05). In vitro, palmitate-BSA triggered endoplasmic reticulum-stress markers, which was accompanied with reduced miR-17-5p and induced thioredoxin interacting protein mRNA in retinal Müller glial cell line (P < 0.05). Palmitate upregulated NLRP3 and IL1β expression in primary Müller cells isolated from wild type. However, using primary Müller cells isolated from thioredoxin interacting protein knock-out mice abolished palmitate-mediated increase in NLRP3 and IL1β. CONCLUSION Our work suggests that targeting endoplasmic reticulum-stress or thioredoxin interacting protein are potential therapeutic strategies for early

  10. The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress.

    PubMed

    Cantero-Recasens, Gerard; Fandos, César; Rubio-Moscardo, Fanny; Valverde, Miguel A; Vicente, Rubén

    2010-01-01

    Alterations of protein folding or Ca(2+) levels within the endoplasmic reticulum (ER) result in the unfolded-protein response (UPR), a process considered as an endogenous inducer of inflammation. Thereby, understanding how genetic factors modify UPR is particularly relevant in chronic inflammatory diseases such as asthma. Here we identified that ORMDL3, the only genetic risk factor recently associated to asthma in a genome wide study, alters ER-mediated Ca(2+) homeostasis and facilitates the UPR. Heterologous expression of human ER-resident transmembrane ORMDL3 protein increased resting cytosolic Ca(2+) levels and reduced ER-mediated Ca(2+) signaling, an effect reverted by co-expression with the sarco-endoplasmic reticulum Ca(2+) pump (SERCA). Increased ORMDL3 expression also promoted stronger activation of UPR transducing molecules and target genes while siRNA-mediated knock-down of endogenous ORMDL3 potentiated ER Ca(2+) release and attenuated the UPR. In conclusion, our findings are consistent with a model in which ORMDL3 binds and inhibits SERCA resulting in a reduced ER Ca(2+) concentration and increased UPR. Thus, we provide a first insight into the molecular mechanism explaining the association of ORMDL3 with proinflammatory diseases.

  11. Effect of lithium chloride on endoplasmic reticulum stress-related PERK/ROCK signaling in a rat model of glaucoma.

    PubMed

    Sun, Xiao-Bo; Lu, Hong-E; Chen, Yuan; Fan, Xiao-Hui; Tong, Bin

    2014-12-01

    Elevated intraocular pressure (IOP) is considered as the major risk factor for the loss of retinal ganglion cells (RGCs) and their axons in glaucoma. Lithium chloride (LiCl) inhibits glycogen synthase kinase-3 beta (GSK-3β) and attends PERK-induced endoplasmic reticulum stress (ERs) transition. PERK is a type I transmembrane protein located in the endoplasmic reticulum. PERK pathway activation takes place in ERs early inhibiting protein synthnesis to protect cell and promote cell survival. Here, we firstly evaluate that LiCl reduced IOP when administered intraperitoneally. After 6 weeks, IOP dropped by around 21.9% in LiCl treated rats. Then we investigated the effects of LiCI on PERK-mediated signaling pathways. LiCl treatment activated PERK and inhibited the expression of ROCK-1 and ROCK-2 in a rat model of glaucoma. Collectively, these results suggest that LiCl reduced the IOP through the phosphorylation of PERK by the regulation of PERK/ROCK signaling in glaucoma rat model.

  12. Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for β cell function.

    PubMed

    Lortz, S; Lenzen, S; Mehmeti, I

    2015-08-01

    Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing.

  13. Redox-assisted regulation of Ca2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5

    PubMed Central

    Ushioda, Ryo; Miyamoto, Akitoshi; Inoue, Michio; Watanabe, Satoshi; Okumura, Masaki; Maegawa, Ken-ichi; Uegaki, Kaiku; Fujii, Shohei; Fukuda, Yasuko; Umitsu, Masataka; Takagi, Junichi; Inaba, Kenji; Mikoshiba, Katsuhiko; Nagata, Kazuhiro

    2016-01-01

    Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER. PMID:27694578

  14. Progressive Depletion of Rough Endoplasmic Reticulum in Epithelial Cells of the Small Intestine in Monosodium Glutamate Mice Model of Obesity

    PubMed Central

    Nakadate, Kazuhiko; Motojima, Kento; Hirakawa, Tomoya; Tanaka-Nakadate, Sawako

    2016-01-01

    Chronic obesity is a known risk factor for metabolic syndrome. However, little is known about pathological changes in the small intestine associated with chronic obesity. This study investigated cellular and subcellular level changes in the small intestine of obese mice. In this study, a mouse model of obesity was established by early postnatal administration of monosodium glutamate. Changes in body weight were monitored, and pathological changes in the small intestine were evaluated using hematoxylin-eosin and Nissl staining and light and electron microscopy. Consequently, obese mice were significantly heavier compared with controls from 9 weeks of age. Villi in the small intestine of obese mice were elongated and thinned. There was reduced hematoxylin staining in the epithelium of the small intestine of obese mice. Electron microscopy revealed a significant decrease in and shortening of rough endoplasmic reticulum in epithelial cells of the small intestine of obese mice compared with normal mice. The decrease in rough endoplasmic reticulum in the small intestine epithelial cells of obese mice indicates that obesity starting in childhood influences various functions of the small intestine, such as protein synthesis, and could impair both the defense mechanism against invasion of pathogenic microbes and nutritional absorption. PMID:27437400

  15. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  16. Progressive Depletion of Rough Endoplasmic Reticulum in Epithelial Cells of the Small Intestine in Monosodium Glutamate Mice Model of Obesity.

    PubMed

    Nakadate, Kazuhiko; Motojima, Kento; Hirakawa, Tomoya; Tanaka-Nakadate, Sawako

    2016-01-01

    Chronic obesity is a known risk factor for metabolic syndrome. However, little is known about pathological changes in the small intestine associated with chronic obesity. This study investigated cellular and subcellular level changes in the small intestine of obese mice. In this study, a mouse model of obesity was established by early postnatal administration of monosodium glutamate. Changes in body weight were monitored, and pathological changes in the small intestine were evaluated using hematoxylin-eosin and Nissl staining and light and electron microscopy. Consequently, obese mice were significantly heavier compared with controls from 9 weeks of age. Villi in the small intestine of obese mice were elongated and thinned. There was reduced hematoxylin staining in the epithelium of the small intestine of obese mice. Electron microscopy revealed a significant decrease in and shortening of rough endoplasmic reticulum in epithelial cells of the small intestine of obese mice compared with normal mice. The decrease in rough endoplasmic reticulum in the small intestine epithelial cells of obese mice indicates that obesity starting in childhood influences various functions of the small intestine, such as protein synthesis, and could impair both the defense mechanism against invasion of pathogenic microbes and nutritional absorption.

  17. Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting Endoplasmic Reticulum (ER) Stress Induced by Disturbed Flow.

    PubMed

    Chung, Jihwa; Kim, Kyoung Hwa; Lee, Seok Cheol; An, Shung Hyun; Kwon, Kihwan

    2015-10-01

    Disturbed blood flow with low-oscillatory shear stress (OSS) is a predominant atherogenic factor leading to dysfunctional endothelial cells (ECs). Recently, it was found that disturbed flow can directly induce endoplasmic reticulum (ER) stress in ECs, thereby playing a critical role in the development and progression of atherosclerosis. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid, has long been used to treat chronic cholestatic liver disease and is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, its role in atherosclerosis remains unexplored. In this study, we demonstrated the anti-atherogenic activity of UDCA via inhibition of disturbed flow-induced ER stress in atherosclerosis. UDCA effectively reduced ER stress, resulting in a reduction in expression of X-box binding protein-1 (XBP-1) and CEBP-homologous protein (CHOP) in ECs. UDCA also inhibits the disturbed flow-induced inflammatory responses such as increases in adhesion molecules, monocyte adhesion to ECs, and apoptosis of ECs. In a mouse model of disturbed flow-induced atherosclerosis, UDCA inhibits atheromatous plaque formation through the alleviation of ER stress and a decrease in adhesion molecules. Taken together, our results revealed that UDCA exerts anti-atherogenic activity in disturbed flow-induced atherosclerosis by inhibiting ER stress and the inflammatory response. This study suggests that UDCA may be a therapeutic agent for prevention or treatment of atherosclerosis.

  18. Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt

    PubMed Central

    Marini, Cecilia; Ravera, Silvia; Buschiazzo, Ambra; Bianchi, Giovanna; Orengo, Anna Maria; Bruno, Silvia; Bottoni, Gianluca; Emionite, Laura; Pastorino, Fabio; Monteverde, Elena; Garaboldi, Lucia; Martella, Roberto; Salani, Barbara; Maggi, Davide; Ponzoni, Mirco; Fais, Franco; Raffaghello, Lizzia; Sambuceti, Gianmario

    2016-01-01

    Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with 18F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that 18F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy. PMID:27121192

  19. Interaction between endoplasmic reticulum stress and caspase 8 activation in retrovirus MoMuLV-ts1-infected astrocytes.

    PubMed

    Liu, Na; Scofield, Virginia L; Qiang, Wenan; Yan, Mingshan; Kuang, Xianghong; Wong, Paul K Y

    2006-05-10

    The murine retrovirus, MoMuLV-ts1, induces progressive paralysis and immune deficiency in FVB/N mice. We have reported previously that ts1 infection causes apoptosis in astrocytes via endoplasmic reticulum (ER) and mitochondrial stress (Liu, N., Kuang, X., Kim, H.T., Stoica, G., Qiang, W., Scofield, V.L., Wong, P.K.Y. Wong. 2004. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in MoMuLV-ts1-induced apoptosis in astrocytes. J. NeuroVirol. 10, 189-198). In the present study, we show that caspase 8 activation in these cells is mediated through ER stress-associated elevation of death receptor DR5 and the C/EBP homologous protein (GADD153/CHOP), an ER stress-initiated transcription factor, rather than through TNFalpha and TNF-R1 interactions on the cell surface. Treatment with Z-IETD-FMK, a specific inhibitor of caspase 8 enzymatic activity, reduced ER stress by two mechanisms: by inhibiting caspase 8 activation, and by preventing cleavage of the ER-associated membrane protein BAP31 into BAP20, which exacerbates the ER stress response. These findings suggest that caspase 8- and ER stress-associated apoptotic pathways are linked in ts1-infected astrocytes.

  20. PKR-like endoplasmic reticulum kinase is necessary for lipogenic activation during HCMV infection.

    PubMed

    Yu, Yongjun; Pierciey, Francis J; Maguire, Tobi G; Alwine, James C

    2013-01-01

    PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for

  1. The (Ca2+ + Mg2+)-stimulated ATPase of the rat parotid endoplasmic reticulum.

    PubMed Central

    Thiyagarajah, P; Lim, S C

    1986-01-01

    A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [( Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane. PMID:2943271

  2. Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury

    PubMed Central

    Sun, Guo-zhu; Gao, Fen-fei; Zhao, Zong-mao; Sun, Hai; Xu, Wei; Wu, Li-wei; He, Yong-chang

    2016-01-01

    Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endoplasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of fluid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These findings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury. PMID:27651773

  3. Endoplasmic reticulum stress is involved in arsenite-induced oxidative injury in rat brain

    SciTech Connect

    Lin, Anya M.Y.; Chao, P.L.; Fang, S.F.; Chi, C.W.; Yang, C.H.

    2007-10-15

    The mechanism underlying sodium arsenite (arsenite)-induced neurotoxicity was investigated in rat brain. Arsenite was locally infused in the substantia nigra (SN) of anesthetized rat. Seven days after infusion, lipid peroxidation in the infused SN was elevated and dopamine level in the ipsilateral striatum was reduced in a concentration-dependent manner (0.3-5 nmol). Furthermore, local infusion of arsenite (5 nmol) decreased GSH content and increased expression of heat shock protein 70 and heme oxygenase-1 in the infused SN. Aggregation of {alpha}-synuclein, a putative pathological protein involved in several CNS neurodegenerative diseases, was elevated in the arsenite-infused SN. From the breakdown pattern of {alpha}-spectrin, both necrosis and apoptosis were involved in the arsenite-induced neurotoxicity. Pyknotic nuclei, cellular shrinkage and cytoplasmic disintegration, indicating necrosis, and TUNEL-positive cells and DNA ladder, indicating apoptosis was observed in the arsenite-infused SN. Arsenite-induced apoptosis was mediated via two different organelle pathways, mitochondria and endoplasmic reticulum (ER). For mitochondrial activation, cytosolic cytochrome c and caspase-3 levels were elevated in the arsenite-infused SN. In ER pathway, arsenite increased activating transcription factor-4, X-box binding protein 1, C/EBP homologues protein (CHOP) and cytosolic immunoglobulin binding protein levels. Moreover, arsenite reduced procaspase 12 levels, an ER-specific enzyme in the infused SN. Taken together, our study suggests that arsenite is capable of inducing oxidative injury in CNS. In addition to mitochondria, ER stress was involved in the arsenite-induced apoptosis. Arsenite-induced neurotoxicity clinically implies a pathophysiological role of arsenite in CNS neurodegeneration.

  4. A novel subfamily of Hsp70s in the endoplasmic reticulum.

    PubMed

    A, R; Tyson, J R; Stirling, C J

    1997-07-01

    The endoplasmic reticulum contains a number of proteins involved in the processing of secretory polypeptides. These include BiP, which is an Hsp70-family member highly conserved throughout evolution. BiP is known to be intimately involved in several aspects of protein biogenesis, but our understanding of these events has been complicated by the recent description of a novel Hsp70-related protein in yeast, Lhauthorp, whose functions overlap with those of BiP. Current indications are that this protein is distributed widely among eukaryotes and that it represents a distinct subfamily of the Hsp70 class of molecular chaperones.

  5. Analysis of site-specific N-glycan remodeling in the endoplasmic reticulum and the Golgi

    PubMed Central

    Hang, Ivan; Lin, Chia-wei; Grant, Oliver C; Fleurkens, Susanna; Villiger, Thomas K; Soos, Miroslav; Morbidelli, Massimo; Woods, Robert J; Gauss, Robert; Aebi, Markus

    2015-01-01

    The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing. PMID:26240167

  6. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.

    PubMed

    Sun, Wenxiang; Li, Yang; Chen, Lu; Chen, Huihui; You, Fuping; Zhou, Xiang; Zhou, Yi; Zhai, Zhonghe; Chen, Danying; Jiang, Zhengfan

    2009-05-26

    We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

  7. FrontiERs: movers and shapers of the higher plant cortical endoplasmic reticulum.

    PubMed

    Sparkes, Imogen; Hawes, Chris; Frigerio, Lorenzo

    2011-12-01

    The endoplasmic reticulum (ER) in higher plants performs many important functions, yet our understanding of how its intricate network shape and dynamics relate to function is very limited. Recent work has begun to unpick key molecular players in the generation of the pleomorphic, highly dynamic ER network structure that pervades the entire cytoplasm. ER movement is acto-myosin dependent. ER shape is dependent on RHD3 (Root Hair Defective 3) and a family of proteins called reticulons. The major challenge that lies ahead is understanding how factors that control ER shape and movement are regulated and how this relates to the numerous functions of the ER.

  8. The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

    PubMed

    Chao de la Barca, Juan Manuel; Simard, Gilles; Amati-Bonneau, Patrizia; Safiedeen, Zainab; Prunier-Mirebeau, Delphine; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Gueguen, Naïg; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Ferré, Marc; Bris, Céline; Kouassi Nzoughet, Judith; Bocca, Cinzia; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Lenaers, Guy; Martinez, M Carmen; Procaccio, Vincent; Reynier, Pascal

    2016-09-15

    Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q(2)cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as

  9. Isolation and fractionation of the endoplasmic reticulum from castor bean (Ricinus communis) endosperm for proteomic analyses.

    PubMed

    Simon, William J; Maltman, Daniel J; Slabas, Antoni R

    2008-01-01

    This chapter describes the preparation and isolation of highly purified endoplasmic reticulum (ER) from the endosperm of developing and germinating castor bean (Ricinus communis) seeds to provide a purified organelle fraction for differential proteomic analyses. The method uses a two-step ultracentrifugation protocol first described by Coughlan (1) and uses sucrose density gradients and a sucrose flotation step to yield purified ER devoid of other contaminating endomembrane material. Using a combination of one dimensional (1D) and two dimensional (2D) gel electrophoresis the complexity and reproducibility of the protein profile of the purified organelle is evaluated prior to detailed proteomic analyses using mass spectrometry based techniques.

  10. The coupling of plasma membrane calcium entry to calcium uptake by endoplasmic reticulum and mitochondria

    PubMed Central

    García-Sancho, Javier

    2014-01-01

    Abstract Cross-talk between organelles and plasma membrane Ca2+ channels is essential for modulation of the cytosolic Ca2+ ([Ca2+]C) signals, but such modulation may differ among cells. In chromaffin cells Ca2+ entry through voltage-operated channels induces calcium release from the endoplasmic reticulum (ER) that amplifies the signal. [Ca2+]C microdomains as high as 20–50 μm are sensed by subplasmalemmal mitochondria, which accumulate large amounts of Ca2+ through the mitochondrial Ca2+ uniporter (MCU). Mitochondria confine the high-Ca2+ microdomains (HCMDs) to beneath the plasma membrane, where exocytosis of secretory vesicles happens. Cell core [Ca2+]C is much smaller (1–2 μm). By acting as a Ca2+ sink, mitochondria stabilise the HCMD in space and time. In non-excitable HEK293 cells, activation of store-operated Ca2+ entry, triggered by ER Ca2+ emptying, also generated subplasmalemmal HCMDs, but, in this case, most of the Ca2+ was taken up by the ER rather than by mitochondria. The smaller size of the [Ca2+]C peak in this case (about 2 μm) may contribute to this outcome, as the sarco-endoplasmic reticulum Ca2+ ATPase has much higher Ca2+ affinity than MCU. It is also possible that the relative positioning of organelles, channels and effectors, as well as cytoskeleton and accessory proteins plays an important role. Key points Cross-talk between organelles and plasma membrane Ca2+ channels modulates cytosolic Ca2+ signals in different ways. In chromaffin cells Ca2+ entry through voltage-operated channels is amplified by Ca2+ release from the endoplasmic reticulum (ER) and generates subplasmalemmal high Ca2+ microdomains (HCMDs) as high as 20–50 μm, which trigger exocytosis. Subplasmalemmal mitochondria take up Ca2+ from HCMDs and avoid progression of the Ca2+ wave towards the cell core. In non-excitable HEK293 cells activation of store-operated Ca2+ entry triggered by ER Ca2+ emptying also generates subplasmalemmal HCMDs of about 2 μm. In this case

  11. The impact of the endoplasmic reticulum protein-folding environment on cancer development.

    PubMed

    Wang, Miao; Kaufman, Randal J

    2014-09-01

    The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells for the storage and regulated release of calcium and as the entrance to the secretory pathway. Protein misfolding in the ER causes accumulation of misfolded proteins (ER stress) and activation of the unfolded protein response (UPR), which has evolved to maintain a productive ER protein-folding environment. Both ER stress and UPR activation are documented in many different human cancers. In this Review, we summarize the impact of ER stress and UPR activation on every aspect of cancer and discuss outstanding questions for which answers will pave the way for therapeutics.

  12. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.

    PubMed

    Hayashi-Nishino, Mitsuko; Fujita, Naonobu; Noda, Takeshi; Yamaguchi, Akihito; Yoshimori, Tamotsu; Yamamoto, Akitsugu

    2009-12-01

    Autophagy is a bulk degradation process in eukaryotic cells and has fundamental roles in cellular homeostasis.The origin and source of autophagosomal membranes are long-standing questions in the field. Using electron microscopy, we show that, in mammalian culture cells, the endoplasmic reticulum (ER) associates with early autophagic structures called isolation membranes (IMs). Overexpression of an Atg4B mutant, which causes defects in autophagosome formation, induces the accumulation of ER-IM complexes. Electron tomography revealed that the ER-IM complex appears as a subdomain of the ER that formed a cradle encircling the IM, and showed that both ER and isolation membranes are interconnected.

  13. Regulation of Protein Secretion Through Controlled Aggregation in the Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Rivera, Victor M.; Wang, Xiurong; Wardwell, Scott; Courage, Nancy L.; Volchuk, Allen; Keenan, Terence; Holt, Dennis A.; Gilman, Michael; Orci, Lelio; Cerasoli, Frank; Rothman, James E.; Clackson, Tim

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

  14. Amyloid-β peptides are generated in mitochondria-associated endoplasmic reticulum membranes.

    PubMed

    Schreiner, Bernadette; Hedskog, Louise; Wiehager, Birgitta; Ankarcrona, Maria

    2015-01-01

    Extracellular aggregates of amyloid-β peptides (Aβ) are a hallmark in Alzheimer's disease (AD) brains. Recent findings suggest that Aβ is generated intracellularly and potential production sites include endosomes and trans-Golgi network. We determined the production of Aβ in subcellular fractions isolated from mouse brain. We found that a considerable amount of Aβ is produced at mitochondria-endoplasmic reticulum (ER) contact sites including outer mitochondrial membrane and mitochondria-associated ER membranes. Enhanced Aβ production at this site may disturb ER, mitochondrial and mitochondria-ER contact site function. This may be one key step in the cascade of events eventually leading to neurodegeneration in AD.

  15. The endoplasmic reticulum exerts control over organelle streaming during cell expansion.

    PubMed

    Stefano, Giovanni; Renna, Luciana; Brandizzi, Federica

    2014-03-01

    Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

  16. Nuclear Receptors Resolve Endoplasmic Reticulum Stress to Improve Hepatic Insulin Resistance

    PubMed Central

    2017-01-01

    Chronic endoplasmic reticulum (ER) stress culminating in proteotoxicity contributes to the development of insulin resistance and progression to type 2 diabetes mellitus. Pharmacologic interventions targeting several different nuclear receptors have emerged as potential treatments for insulin resistance. The mechanistic basis for these antidiabetic effects has primarily been attributed to multiple metabolic and inflammatory functions. Here we review recent advances in our understanding of the association of ER stress with insulin resistance and the role of nuclear receptors in promoting ER stress resolution and improving insulin resistance in the liver. PMID:28236381

  17. [From endoplasmic reticulum to Golgi apparatus: a secretory pathway controlled by signal molecules].

    PubMed

    Wang, Jiasheng; Luo, Jianhong; Zhang, Xiaomin

    2013-07-01

    Protein transport from endoplasmic reticulum (ER) to Golgi apparatus has long been known to be a central process for protein quality control and sorting. Recent studies have revealed that a large number of signal molecules are involved in regulation of membrane trafficking through ER, ER-Golgi intermediate compartment and Golgi apparatus. These molecules can significantly change the transport rate of proteins by regulating vesicle budding and fusion. Protein transport from ER to Golgi apparatus is not only controlled by signal pathways triggered from outside the cell, it is also regulated by feedback signals from the transport pathway.

  18. Oncogenic and oncosuppressive signal transduction at mitochondria-associated endoplasmic reticulum membranes

    PubMed Central

    Marchi, Saverio; Giorgi, Carlotta; Oparka, Monika; Duszynski, Jerzy; Wieckowski, Mariusz R; Pinton, Paolo

    2014-01-01

    The different mechanisms employed by proto-oncogenes and tumor suppressors to regulate cell death pathways are strictly linked to their localization. In addition to the canonical control of apoptosis at a transcriptional/nuclear level, intracellular zones are emerging as pivotal sites for the activities of several proapoptotic and antiapoptotic factors. Here, we review the function of the endoplasmic reticulum-mitochondria interface as a primary platform for decoding danger signals as well as a structural accommodation for several regulator or effector proteins. PMID:27308328

  19. BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress

    PubMed Central

    Carpio, Marcos A.; Michaud, Michael; Zhou, Wenping; Fisher, Jill K.; Walensky, Loren D.; Katz, Samuel G.

    2015-01-01

    B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a BCL-2 family protein with high homology to the multidomain proapoptotic proteins BAX and BAK, yet Bok−/− and even Bax−/−Bok−/− and Bak−/−Bok−/− mice were reported to have no overt phenotype or apoptotic defects in response to a host of classical stress stimuli. These surprising findings were interpreted to reflect functional compensation among the BAX, BAK, and BOK proteins. However, BOK cannot compensate for the severe apoptotic defects of Bax−/−Bak−/− mice despite its widespread expression. Here, we independently developed Bok−/− mice and found that Bok−/− cells are selectively defective in their response to endoplasmic reticulum (ER) stress stimuli, consistent with the predominant subcellular localization of BOK at the ER. Whereas Bok−/− mouse embryonic fibroblasts exposed to thapsigargin, A23187, brefeldin A, DTT, geldanamycin, or bortezomib manifested reduced activation of the mitochondrial apoptotic pathway, the death response to other stimuli such as etoposide, staurosporine, or UV remained fully intact. Multiple organs in Bok−/− mice exhibited resistance to thapsigargin-induced apoptosis in vivo. Although the ER stress agents activated the unfolded protein response, both ATF4 and CHOP activation were diminished in Bok−/− cells and mice. Importantly, BAX and BAK were unable to compensate for the defective apoptotic response to ER stress observed in SV40-transformed and primary Bok−/− cells, and in vivo. These findings support a selective and distinguishing role for BOK in regulating the apoptotic response to ER stress, revealing—to our knowledge—the first bona fide apoptotic defect linked to Bok deletion. PMID:26015568

  20. Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells

    PubMed Central

    Yi, Wei; Yang, Yang; Zhao, Dajun; Yang, Honggang; Geng, Ting; Xing, Jianzhou; Zhang, Yu; Tan, Songtao; Yi, Dinghua

    2014-01-01

    Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine (sICAM1, sE-selectin, MCP-1 and IL-8) production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK). EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin (THA, an ERS inducer) attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS. PMID:25470819

  1. High-Density Lipoprotein Prevents Endoplasmic Reticulum Stress-Induced Downregulation of Liver LOX-1 Expression.

    PubMed

    Hong, Dan; Li, Ling-Fang; Gao, Hai-Chao; Wang, Xiang; Li, Chuan-Chang; Luo, Ying; Bai, Yong-Ping; Zhang, Guo-Gang

    2015-01-01

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a specific cell-surface receptor for oxidized-low-density lipoprotein (ox-LDL). The impact of high-density lipoprotein (HDL) on endoplasmic reticulum (ER) stress-mediated alteration of the LOX-1 level in hepatocytes remains unclear. We aimed to investigate the impact on LOX-1 expression by tunicamycin (TM)-induced ER stress and to determine the effect of HDL on TM-affected LOX-1 expression in hepatic L02 cells. Overexpression or silencing of related cellular genes was conducted in TM-treated cells. mRNA expression was evaluated using real-time polymerase chain reaction (PCR). Protein expression was analyzed by western blot and immunocytochemistry. Lipid uptake was examined by DiI-ox-LDL, followed by flow cytometric analysis. The results showed that TM induced the upregulation of ER chaperone GRP78, downregulation of LOX-1 expression, and lipid uptake. Knock down of IRE1 or XBP-1 effectively restored LOX-1 expression and improved lipid uptake in TM-treated cells. HDL treatment prevented the negative impact on LOX-1 expression and lipid uptake induced by TM. Additionally, 1-10 μg/mL HDL significantly reduced the GRP78, IRE1, and XBP-1 expression levels in TM-treated cells. Our findings reveal that HDL could prevent the TM-induced reduction of LOX-1 expression via inhibiting the IRE1/XBP-1 pathway, suggesting a new mechanism for beneficial roles of HDL in improving lipid metabolism.

  2. The Batten disease gene CLN3 confers resistance to endoplasmic reticulum stress induced by tunicamycin

    SciTech Connect

    Wu, Dan; Liu, Jing; Wu, Baiyan; Tu, Bo; Zhu, Weiguo; Luo, Jianyuan

    2014-04-25

    Highlights: • The work reveals a protective properties of CLN3 towards TM-induced apoptosis. • CLN3 regulates expression of the GRP78 and the CHOP in response to the ER stress. • CLN3 plays a specific role in the ERS response. - Abstract: Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.

  3. Neuronal calcium wave propagation varies with changes in endoplasmic reticulum parameters: a computer model

    PubMed Central

    Neymotin, Samuel A.; McDougal, Robert A.; Sherif, Mohamed A.; Fall, Christopher P.; Hines, Michael L.; Lytton, William W.

    2015-01-01

    Calcium (Ca2+) waves provide a complement to neuronal electrical signaling, forming a key part of a neuron’s second messenger system. We developed a reaction-diffusion model of an apical dendrite with diffusible inositol triphosphate (IP3), diffusible Ca2+, IP3 receptors (IP3Rs), endoplasmic reticulum (ER) Ca2+ leak, and ER pump (SERCA) on ER. Ca2+ is released from ER stores via IP3Rs upon binding of IP3 and Ca2+. This results in Ca2+-induced-Ca2+-release (CICR) and increases Ca2+ spread. At least two modes of Ca2+ wave spread have been suggested: a continuous mode based on presumed relative homogeneity of ER within the cell; and a pseudo-saltatory model where Ca2+ regeneration occurs at discrete points with diffusion between them. We compared the effects of three patterns of hypothesized IP3R distribution: 1. continuous homogeneous ER, 2. hotspots with increased IP3R density (IP3R hotspots), 3. areas of increased ER density (ER stacks). All three modes produced Ca2+ waves with velocities similar to those measured in vitro (~50–90µm /sec). Continuous ER showed high sensitivity to IP3R density increases, with time to onset reduced and speed increased. Increases in SERCA density resulted in opposite effects. The measures were sensitive to changes in density and spacing of IP3R hotspots and stacks. Increasing the apparent diffusion coefficient of Ca2+ substantially increased wave speed. An extended electrochemical model, including voltage gated calcium channels and AMPA synapses, demonstrated that membrane priming via AMPA stimulation enhances subsequent Ca2+ wave amplitude and duration. Our modeling suggests that pharmacological targeting of IP3Rs and SERCA could allow modulation of Ca2+ wave propagation in diseases where Ca2+ dysregulation has been implicated. PMID:25734493

  4. Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

    PubMed Central

    Santi-Rocca, Julien; Smith, Sherri; Weber, Christian; Pineda, Erika; Hon, Chung-Chau; Saavedra, Emma; Olivos-García, Alfonso; Rousseau, Sandrine; Dillies, Marie-Agnès; Coppée, Jean-Yves; Guillén, Nancy

    2012-01-01

    The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR). The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO) triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO) and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i) dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii) induction of DNA repair and redox gene expression and iii) up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis. PMID:22384074

  5. Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis

    PubMed Central

    Moreira, Andrea Janz; Ordoñez, Raquel; Cerski, Carlos Thadeu; Picada, Jaqueline Nascimento; García-Palomo, Andrés; Marroni, Norma Possa; Mauriz, Jose L.; González-Gallego, Javier

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN) 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP) ribose polymerase (PARP) cleavage, and Bcl-associated X protein (Bax)/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6), C/EBP-homologous protein (CHOP) and immunoglobulin heavy chain-binding protein (BiP), while cyclooxygenase (COX)-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC. PMID:26656265

  6. Endoplasmic Reticulum Ca(2+) Handling and Apoptotic Resistance in Tumor-Derived Endothelial Colony Forming Cells.

    PubMed

    Poletto, Valentina; Dragoni, Silvia; Lim, Dmitry; Biggiogera, Marco; Aronica, Adele; Cinelli, Mariapia; De Luca, Antonio; Rosti, Vittorio; Porta, Camillo; Guerra, Germano; Moccia, Francesco

    2016-10-01

    Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc.

  7. Activation of Toll-like Receptor 4 (TLR4) Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress*

    PubMed Central

    Okla, Meshail; Wang, Wei; Kang, Inhae; Pashaj, Anjeza; Carr, Timothy; Chung, Soonkyu

    2015-01-01

    Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study we hypothesized that Toll-like receptor 4 (TLR4) activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by a high fat diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of endoplasmic reticulum (ER) stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced up-regulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data indicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective brown adipose tissue activation. PMID:26370079

  8. Asbestos-induced alveolar epithelial cell apoptosis. The role of endoplasmic reticulum stress response.

    PubMed

    Kamp, David W; Liu, Gang; Cheresh, Paul; Kim, Seok-Jo; Mueller, Amanda; Lam, Anna P; Trejo, Humberto; Williams, David; Tulasiram, Sandhya; Baker, Margaret; Ridge, Karen; Chandel, Navdeep S; Beri, Rohinee

    2013-12-01

    Asbestos exposure results in pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) apoptosis is important in the development of pulmonary fibrosis after exposure to an array of toxins, including asbestos. An endoplasmic reticulum (ER) stress response and mitochondria-regulated (intrinsic) apoptosis occur in AECs of patients with idiopathic pulmonary fibrosis, a disease with similarities to asbestosis. Asbestos induces AEC intrinsic apoptosis, but the role of the ER is unclear. The objective of this study was to determine whether asbestos causes an AEC ER stress response that promotes apoptosis. Using human A549 and rat primary isolated alveolar type II cells, amosite asbestos fibers increased AEC mRNA and protein expression of ER stress proteins involved in the unfolded protein response, such as inositol-requiring kinase (IRE) 1 and X-box-binding protein-1, as well as ER Ca²(2+) release ,as assessed by a FURA-2 assay. Eukarion-134, a superoxide dismutase/catalase mimetic, as well as overexpression of Bcl-XL in A549 cells each attenuate asbestos-induced AEC ER stress (IRE-1 and X-box-binding protein-1 protein expression; ER Ca²(2+) release) and apoptosis. Thapsigargin, a known ER stress inducer, augments AEC apoptosis, and eukarion-134 or Bcl-XL overexpression are protective. Finally, 4-phenylbutyric acid, a chemical chaperone that attenuates ER stress, blocks asbestos- and thapsigargin-induced AEC IRE-1 protein expression, but does not reduce ER Ca²(2+) release or apoptosis. These results show that asbestos triggers an AEC ER stress response and subsequent intrinsic apoptosis that is mediated in part by ER Ca²(2+) release.

  9. Nongenomic STAT5-dependent effects on Golgi apparatus and endoplasmic reticulum structure and function.

    PubMed

    Lee, Jason E; Yang, Yang-Ming; Liang, Feng-Xia; Gough, Daniel J; Levy, David E; Sehgal, Pravin B

    2012-03-01

    We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.

  10. PFKFB3 modulates glycolytic metabolism and alleviates endoplasmic reticulum stress in human osteoarthritis cartilage.

    PubMed

    Qu, Jining; Lu, Daigang; Guo, Hua; Miao, Wusheng; Wu, Ge; Zhou, Meifen

    2016-03-01

    Glycolytic disorder has been demonstrated to be a major cause of osteoarthritis (OA) and chondrocyte dysfunction. The present work aimed to investigate the expression and role of the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in OA cartilage. It was found that PFKFB3 expression was down-regulated in human OA cartilage tissues and in tumour necrosis factor (TNF)-α- or interleukin (IL)-1β-stimulated human chondrocytes. The glycolytic metabolism appeared as glucose utilization and adenosine triphosphate (ATP) generation, and lactate production was stunted in OA cartilage. However, the impaired glycolytic process in OA cartilage was improved by PFKFB3 overexpression, which was confirmed in TNF-α- or IL-1β-treated chondrocytes. Furthermore, the expressions of endoplasmic reticulum (ER) stress-associated genes including PERK, ATF3, IRE1, phosphorylated eIF2α (p-eIF2α) and MMP13 were enhanced in OA cartilage explants, while they were decreased by AdPFKFB3 transfection. PFKFB3 also modulated the expressions of PERK, ATF3, IRE1, p-eIF2α and MMP13 in tunicamycin-exposed chondrocytes. Additionally, PFKFB3 improved the cell viability of OA cartilage explants and chondrocytes through the PI3K/Akt/C/EBP homologous protein (CHOP) signalling pathway. The transfection of AdPFKFB3 also significantly reduced caspase 3 activation and promoted aggrecan and type II collagen expressions in OA cartilage explants and chondrocytes. In all, this study characterizes a novel role of PFKFB3 in glycolytic metabolism and ER stress of OA cartilage explants and chondrocytes. The study might provide a potential target for OA prevention or therapy.

  11. Asbestos-induced disruption of calcium homeostasis induces endoplasmic reticulum stress in macrophages.

    PubMed

    Ryan, Alan J; Larson-Casey, Jennifer L; He, Chao; Murthy, Shuhba; Carter, A Brent

    2014-11-28

    Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development.

  12. Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells.

    PubMed

    Quan, Xiaojuan; Wang, Juyan; Liang, Chunlian; Zheng, Huadong; Zhang, Lin

    2015-08-07

    The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide. Melatonin possesses various beneficial metabolic actions, decreased levels of which may accelerate T2D. Endoplasmic reticulum stress (ERS) has been linked to insulin resistance in multiple tissues, but the role of melatonin on ERS and insulin resistance in skeletal muscle has not yet been investigated. In this study, the results showed that tunicamycin decreased insulin-stimulated Akt phosphorylation, but promoted the phosphorylation of protein kinase R-like ER protein kinase (PERK) time-dependently in C2C12 cells. Consistently, ERS gene markers, including binding immunoglobulin protein (BIP)/glucose regulated protein 78 (GRP78) expression and the splicing of X box binding protein 1 (XBP-1), were activated by tunicamycin time-dependently. Interestingly, melatonin pretreatment reversed the elevated PERK phosphorylation, as well as the activation of Bip expression and XBP-1 splicing, and prevented the inhibitory effect of tunicamycin on Akt phosphorylation. In addition, the insulin-provoked glucose transport was reduced by tunicamycin, and then promoted by melatonin pretreatment. A strong phosphorylation of inositol-requiring enzyme 1 (IRE-1), c-JUN NH2-terminal kinase (JNK), and insulin receptor substrate 1 (IRS-1) serine, and simultaneously, a dramatic decrease of IRS-1 tyrosine phosphorylation were observed in the presence of tunicamycin, leading to a blockade of insulin signaling, which was reversed by melatonin pretreatment. Furthermore, luzindole pretreatment acted inversely with melatonin action on glucose uptake and insulin signaling. Therefore, these results demonstrated that melatonin pretreatment inhibited the activated role of tunicamycin on ERS and insulin resistance through melatonin receptor-mediated IRE-1/JNK/IRS-1 insulin signaling in skeletal muscle cells.

  13. The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane

    PubMed Central

    Honvo-Houéto, Edith; Henry, Céline; Chat, Sophie; Layani, Sarah; Truchet, Sandrine

    2016-01-01

    During lactation, mammary epithelial cells secrete huge amounts of milk from their apical side. The current view is that caseins are secreted by exocytosis, whereas milk fat globules are released by budding, enwrapped by the plasma membrane. Owing to the number and large size of milk fat globules, the membrane surface needed for their release might exceed that of the apical plasma membrane. A large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globule membranes was used to decipher the cellular origins of the milk fat globule membrane. Surprisingly, differential analysis of protein profiles of these two organelles strongly suggest that, in addition to the plasma membrane, the endoplasmic reticulum and the secretory vesicles contribute to the milk fat globule membrane. Analysis of membrane-associated and raft microdomain proteins reinforces this possibility and also points to a role for lipid rafts in milk product secretion. Our results provide evidence for a significant contribution of the endoplasmic reticulum to the milk fat globule membrane and a role for SNAREs in membrane dynamics during milk secretion. These novel aspects point to a more complex model for milk secretion than currently envisioned. PMID:27535430

  14. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology.

    PubMed

    Guerriero, Christopher J; Brodsky, Jeffrey L

    2012-04-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding "problem," as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.

  15. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

    PubMed Central

    Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

  16. p53 and Ca(2+) signaling from the endoplasmic reticulum: partners in anti-cancer therapies.

    PubMed

    Bittremieux, Mart; Bultynck, Geert

    2015-01-01

    Ca(2+) transfer from the endoplasmic reticulum (ER) to the mitochondria critically controls cell survival and cell death decisions. Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca(2+) levels by enhancing sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity, ultimately promoting mitochondrial Ca(2+) overload. The boosting of "toxic" Ca(2+) signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca(2+) transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca(2+) uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca(2+) measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca(2+) signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.

  17. Sphingosine-1-phosphate Phosphatase 2 Regulates Pancreatic Islet β-Cell Endoplasmic Reticulum Stress and Proliferation.

    PubMed

    Taguchi, Yoshimitsu; Allende, Maria L; Mizukami, Hiroki; Cook, Emily K; Gavrilova, Oksana; Tuymetova, Galina; Clarke, Benjamin A; Chen, Weiping; Olivera, Ana; Proia, Richard L

    2016-06-03

    Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive β-cell proliferation that was demonstrated after treatment with either a high-fat diet or the β-cell-specific toxin, streptozotocin. Importantly, β-cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with β-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes β-cell endoplasmic reticulum stress, which is a known cause of β-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes.

  18. Photodynamic action of porphyrin on Ca2+ influx in endoplasmic reticulum: a comparison with mitochondria.

    PubMed Central

    Ricchelli, F; Barbato, P; Milani, M; Gobbo, S; Salet, C; Moreno, G

    1999-01-01

    We have studied the distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) in mitochondria and endoplasmic reticulum after isolation from rat liver. The photosensitizing efficiency of porphyrin on the Ca2+ influx function of microsomes has been compared with that obtained on Ca2+ uptake in mitochondria. HP and PP are accumulated in microsomes to a greater extent than in mitochondria, both porphyrins binding to membrane protein sites. The Ca2+ influx functions of mitochondria and microsomes, before and after irradiation in the presence of HP or PP, were studied by following the changes in the free Ca2+ concentration in the medium as revealed by the variations in fluorescence intensity of the Ca2+ indicator Calcium Green-1. For the same amount of incorporated porphyrin, the Ca2+ influx function of microsomes is degraded by irradiation more rapidly than that of mitochondria. The protective effect of dithiothreitol suggests that thiol groups in the Ca2+-transporting enzyme are the preferential targets of the photodynamic effect. These results suggest that intracellular Ca2+ movements are altered primarily by the endoplasmic reticulum rather than by mitochondrial damage, in good agreement with other observations made in porphyrin-loaded irradiated cells. PMID:9931319

  19. Endoplasmic reticulum stress plays critical role in brain damage after chronic intermittent hypoxia in growing rats.

    PubMed

    Cai, Xiao-Hong; Li, Xiu-Cui; Jin, Sheng-Wei; Liang, Dong-Shi; Wen, Zheng-Wang; Cao, Hong-Chao; Mei, Hong-Fang; Wu, Ying; Lin, Zhong-Dong; Wang, Liang-Xing

    2014-07-01

    Obstructive sleep apnea hypopnea syndrome (OSAHS) in children is associated with multiple system morbidities. Cognitive dysfunction as a result of central nervous system complication has been reported in children with OSAHS. However, the underlying mechanisms are poorly understood. Endoplasmic reticulum stress (ERS)-related apoptosis plays an important role in various diseases of the central nervous system, but very little is known about the role of ERS in mediating pathophysiological reactions to cognitive dysfunction in OSAHS. Chronic intermittent hypoxia (CIH) exposures, modeling OSAHS, across 2 and 4weeks in growing rats made more reference memory errors, working memory errors and total memory errors in the 8-Arm radial maze task, increased significantly TUNEL positive cells, upregulated the unfolded protein response in the hippocampus and prefrontal cortex as evidenced by increased phosphorylation of PKR-like endoplasmic reticulum kinase, inositol-requiring enzyme l and some downstream products. A selective inhibitor of eukaryotic initiation factor-2a dephosphorylation, salubrinal, prevented C/EBP-homologous protein activation in the hippocampus and prefrontal cortex throughout hypoxia/reoxygenation exposure. Our findings suggest that ERS mediated cell apoptosis may be one of the underlying mechanisms of cognitive dysfunction in OSAHS children. Further, a specific ERS inhibitor Salubrinal should be tested for neuroprotection against CIH-induced injury.

  20. Carbon monoxide-releasing molecules reverse leptin resistance induced by endoplasmic reticulum stress.

    PubMed

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Kim, Seul-Ki; Uddin, Md Jamal; Rhew, Hyunyul; Kim, Taeksang; Ryter, Stefan W; Chung, Hun Taeg

    2013-04-01

    Leptin, a circulating hormone, regulates food intake and body weight. While leptin resistance represents a major cause of obesity, the underlying mechanisms remain unclear. Endoplasmic reticulum (ER) stress can contribute to leptin resistance. Carbon monoxide (CO), a gaseous molecule, exerts antiapoptotic and anti-inflammatory effects in animal models of tissue injury. We hypothesized that CO could inhibit leptin resistance during ER stress. Thapsigargin or tunicamycin was used to induce ER stress in human cells expressing the leptin receptor. These agents markedly inhibited leptin-induced STAT3 phosphorylation, confirming that ER stress induces leptin resistance. The CO-releasing molecule CORM-2 blocked the ER stress-dependent inhibition of leptin-induced STAT3 phosphorylation. CORM-2 treatment induced the phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK), and eukaryotic translation initiation factor-2α and enhanced PERK phosphorylation during ER stress. Furthermore, CORM-2 inhibited X-box binding protein-1 expression, activating transcription factor-6 cleavage, and inositol-requiring enzyme (IRE)1α phosphorylation induced by ER stress. IRE1α knockdown rescued leptin resistance, whereas PERK knockdown blocked CO-dependent regulation of IRE1α. In vivo, CO inhalation normalized body weight in animals fed high-fat diets. Furthermore, CO modulated ER stress pathways and rescued leptin resistance in vivo. In conclusion, the pathological mechanism of leptin resistance may be ameliorated by the pharmacological application of CO.

  1. Oxidative stress contributes to autophagy induction in response to endoplasmic reticulum stress in Chlamydomonas reinhardtii.

    PubMed

    Pérez-Martín, Marta; Pérez-Pérez, María Esther; Lemaire, Stéphane D; Crespo, José L

    2014-10-01

    The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) results in the activation of stress responses, such as the unfolded protein response or the catabolic process of autophagy to ultimately recover cellular homeostasis. ER stress also promotes the production of reactive oxygen species, which play an important role in autophagy regulation. However, it remains unknown whether reactive oxygen species are involved in ER stress-induced autophagy. In this study, we provide evidence connecting redox imbalance caused by ER stress and autophagy activation in the model unicellular green alga Chlamydomonas reinhardtii. Treatment of C. reinhardtii cells with the ER stressors tunicamycin or dithiothreitol resulted in up-regulation of the expression of genes encoding ER resident endoplasmic reticulum oxidoreductin1 oxidoreductase and protein disulfide isomerases. ER stress also triggered autophagy in C. reinhardtii based on the protein abundance, lipidation, cellular distribution, and mRNA levels of the autophagy marker ATG8. Moreover, increases in the oxidation of the glutathione pool and the expression of oxidative stress-related genes were detected in tunicamycin-treated cells. Our results revealed that the antioxidant glutathione partially suppressed ER stress-induced autophagy and decreased the toxicity of tunicamycin, suggesting that oxidative stress participates in the control of autophagy in response to ER stress in C. reinhardtii In close agreement, we also found that autophagy activation by tunicamycin was more pronounced in the C. reinhardtii sor1 mutant, which shows increased expression of oxidative stress-related genes.

  2. EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells.

    PubMed

    Gadalla, Salah-Eldin; Öjemalm, Karin; Vasquez, Patricia Lara; Nilsson, Ingmarie; Ericsson, Christer; Zhao, Jian; Nistér, Monica

    2013-09-20

    Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell "marker" and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

  3. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  4. Insulin Dissociates the Effects of Liver X Receptor on Lipogenesis, Endoplasmic Reticulum Stress, and Inflammation*

    PubMed Central

    Sun, Xiaowei; Haas, Mary E.; Miao, Ji; Mehta, Abhiruchi; Graham, Mark J.; Crooke, Rosanne M.; de Barros, Jean-Paul Pais; Wang, Jian-Guo; Aikawa, Masanori; Masson, David; Biddinger, Sudha B.

    2016-01-01

    Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor liver X receptor (LXR) is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knock down Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knock-out of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation, and ER stress from the negative effects on lipogenesis. PMID:26511317

  5. Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

    PubMed Central

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida; Sanvito, Rossella; Magni, Fulvio; Coccetti, Paola; Rocchetti, Marcella; Nielsen, Jens; Alberghina, Lilia; Vanoni, Marco

    2016-01-01

    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage. PMID:27305947

  6. Endoplasmic reticulum stress-mediated hippocampal neuron apoptosis involved in diabetic cognitive impairment.

    PubMed

    Zhang, Xiaoming; Xu, Linhao; He, Daqiang; Ling, Shucai

    2013-01-01

    Poor management of DM causes cognitive impairment while the mechanism is still unconfirmed. The aim of the present study was to investigate the activation of C/EBP Homology Protein (CHOP), the prominent mediator of the endoplasmic reticulum (ER) stress-induced apoptosis under hyperglycemia. We employed streptozotocin- (STZ-) induced diabetic rats to explore the ability of learning and memory by the Morris water maze test. The ultrastructure of hippocampus in diabetic rats and cultured neurons in high glucose medium were observed by transmission electron microscopy and scanning electron microscopy. TUNEL staining was also performed to assess apoptotic cells while the expression of CHOP was assayed by immunohistochemistry and Western blot assay in these hippocampal neurons. Six weeks after diabetes induction, the escape latency increased and the average frequency in finding the platform decreased in diabetic rats (P < 0.05). The morphology of neuron and synaptic structure was impaired; the number of TUNEL-positive cells and the expression of CHOP in hippocampus of diabetic rats and high glucose medium cultured neurons were markedly altered (P < 0.05). The present results suggested that the CHOP-dependent endoplasmic reticulum (ER) stress-mediated apoptosis may be involved in hyperglycemia-induced hippocampal synapses and neurons impairment and promote the diabetic cognitive impairment.

  7. Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury

    PubMed Central

    Folch-Puy, Emma; Panisello, Arnau; Oliva, Joan; Lopez, Alexandre; Castro Benítez, Carlos; Adam, René; Roselló-Catafau, Joan

    2016-01-01

    The endoplasmic reticulum (ER) is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS). This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR), which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI) of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes. PMID:27231901

  8. Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum.

    PubMed

    Mund, Thomas; Gewies, Andreas; Schoenfeld, Nicole; Bauer, Manuel K A; Grimm, Stefan

    2003-04-01

    We have isolated Spike, a novel and evolutionary conserved BH3-only protein. BH3-only proteins constitute a family of apoptosis inducers that mediate proapoptotic signals. In contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Furthermore, unlike the known BH3-only proteins, Spike could not interact with all tested Bcl-2 family members, despite its BH3 domain being necessary for cell killing. Our findings indicate that Spike is localized to the endoplasmic reticulum. The endoplasmic reticulum is an organelle that has only recently been implicated in regulation of apoptosis. At this locale, Spike interacts with Bap31, an adaptor protein for pro-caspase-8 and Bcl-XL. In doing so, Spike is able to inhibit the formation of a complex between Bap31 and the antiapoptotic Bcl-XL protein. Furthermore, Spike transmits the signal of specific death receptors. Its down-regulation in certain tumors suggests that Spike may also play a role in tumorigenesis. Our findings add new insight for how BH3-only and antiapoptotic Bcl-2 proteins regulate cell death.

  9. Erp29 Attenuates Cigarette Smoke Extract–Induced Endoplasmic Reticulum Stress and Mitigates Tight Junction Damage in Retinal Pigment Epithelial Cells

    PubMed Central

    Huang, Chuangxin; Wang, Joshua J.; Jing, Guangjun; Li, Junhua; Jin, Chenjin; Yu, Qiang; Falkowski, Marek W.; Zhang, Sarah X.

    2015-01-01

    Purpose Endoplasmic reticulum protein 29 (ERp29) is a novel chaperone that was recently found decreased in human retinas with AMD. Herein, we examined the effect of ERp29 on cigarette smoke–induced RPE apoptosis and tight junction disruption. Methods Cultured human RPE (HRPE) cells (ARPE-19) or mouse RPE eyecup explants were exposed to cigarette smoke extract (CSE) for short (up to 24 hours) or long (up to 3 weeks) periods. Expression of ERp29 was up- and downregulated by adenovirus and siRNA, respectively. Endoplasmic reticulum stress markers, apoptosis, and cell death, the expression and distribution of tight junction protein ZO-1, transepithelial electrical resistance (TEER), and F-actin expression were examined. Results Endoplasmic reticulum protein 29 was significantly increased by short-term exposure to CSE in ARPE-19 cells or eyecup explants but was reduced after 3-week exposure. Overexpression of ERp29 increased the levels of GRP78, p58IPK, and Nrf-2, while reducing p-eIF2α and C/EBP homologous protein (CHOP), and protected RPE cells from CSE-induced apoptosis. In contrast, knockdown of ERp29 decreased the levels of p58IPK and Nrf2, but increased p-eIF2α and CHOP and exacerbated CSE-triggered cell death. In addition, overexpression of ERp29 attenuated CSE-induced reduction in ZO-1 and enhanced the RPE barrier function, as measured by TEER. Knockdown of ERp29 decreased the level of ZO-1 protein. These effects were associated with changes in the expression of cytoskeleton F-actin. Conclusions Endoplasmic reticulum protein 29 attenuates CSE-induced ER stress and enhances cell viability and barrier integrity of RPE cells, and therefore may act as a protective mechanism for RPE survival and activity. PMID:26431474

  10. Severe Injury Is Associated With Insulin Resistance, Endoplasmic Reticulum Stress Response, and Unfolded Protein Response

    PubMed Central

    Jeschke, Marc G.; Finnerty, Celeste C.; Herndon, David N.; Song, Juquan; Boehning, Darren; Tompkins, Ronald G.; Baker, Henry V.; Gauglitz, Gerd G.

    2012-01-01

    Objective We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling. Background Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR. Methods Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance–related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn. Results Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn. Conclusion Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time. PMID:22241293

  11. Calcium Efflux From the Endoplasmic Reticulum Leads to β-Cell Death

    PubMed Central

    Hara, Takashi; Mahadevan, Jana; Kanekura, Kohsuke; Hara, Mariko; Lu, Simin

    2014-01-01

    It has been established that intracellular calcium homeostasis is critical for survival and function of pancreatic β-cells. However, the role of endoplasmic reticulum (ER) calcium homeostasis in β-cell survival and death is not clear. Here we show that ER calcium depletion plays a critical role in β-cell death. Various pathological conditions associated with β-cell death, including ER stress, oxidative stress, palmitate, and chronic high glucose, decreased ER calcium levels and sarcoendoplasmic reticulum Ca2+-ATPase 2b expression, leading to β-cell death. Ectopic expression of mutant insulin and genetic ablation of WFS1, a causative gene for Wolfram syndrome, also decreased ER calcium levels and induced β-cell death. Hyperactivation of calpain-2, a calcium-dependent proapoptotic protease, was detected in β-cells undergoing ER calcium depletion. Ectopic expression of sarcoendoplasmic reticulum Ca2+-ATPase 2b, as well as pioglitazone and rapamycin treatment, could prevent calcium efflux from the ER and mitigate β-cell death under various stress conditions. Our results reveal a critical role of ER calcium depletion in β-cell death and indicate that identification of pathways and chemical compounds restoring ER calcium levels will lead to novel therapeutic modalities and pharmacological interventions for type 1 and type 2 diabetes and other ER-related diseases including Wolfram syndrome. PMID:24424032

  12. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.

  13. Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes.

    PubMed

    Guan, Cuiping; Xu, Wen; Hong, Weisong; Zhou, Miaoni; Lin, Fuquan; Fu, Lifang; Liu, Dongyin; Xu, Aie

    2015-06-01

    Swollen endoplasmic reticulum (ER) is commonly observed in the melanocytes of vitiligo patients; however, the cause and proteins involved in this remain to be elucidated. Oxidative stress has been reported to be involved in the pathogenesis of vitiligo and previous studies have demonstrated that hydrogen peroxide (H2O2) induced melanocyte apoptosis, whereas quercetin exhibited cytoprotective activities against the effects of H2O2. The aim of the present study was to further investigate the role of H2O2 in the ER of melanocytes as well as its role in the export of tyrosinase from ER; in addition, the present study aimed to determine the mechanism by which quercetin protects against the effects of H2O2. The results demonstrated that melanocyte cells treated with H2O2 presented with swollen ER; however, a normal ER configuration was observed in untreated cells as well as quercetin/H2O2‑treated cells. Furthermore, H2O2 inhibited tyrosinase export from the ER and decreased expression levels of tyrosinase; however, quercetin was found to attenuate the effects induced by H2O2. In conclusion, the results of the present study confirmed the hypothesis that H2O2 induced ER dilation and hindered functional tyrosinase export from the ER of melanocytes. It was also found that quercetin significantly weakened these effects mediated by H2O2, therefore it may have the potential for use in the treatment of vitiligo.

  14. Glutamine treatment attenuates endoplasmic reticulum stress and apoptosis in TNBS-induced colitis.

    PubMed

    Crespo, Irene; San-Miguel, Beatriz; Prause, Carolina; Marroni, Norma; Cuevas, María J; González-Gallego, Javier; Tuñón, María J

    2012-01-01

    Endoplasmic reticulum (ER) stress and apoptotic cell death play an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). We aimed to explore the potential of glutamine to reduce ER stress and apoptosis in a rat model of experimental IBD. Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/dL) was given by rectal route daily for 2 d or 7 d. Both oxidative stress (TBARS concentration and oxidised/reduced glutathione ratio) and ER stress markers (CHOP, BiP, calpain-1 and caspase-12 expression) increased significantly within 48 h of TNBS instillation, and glutamine attenuated the extent of the changes. Glutamine also inhibited the significant increases of ATF6, ATF4 and spliced XBP-1 mRNA levels induced by TNBS instillation. TNBS-colitis resulted in a significant increase in p53 and cytochrome c expression, and a reduced Bcl-xL expression and Bax/Bcl-2 ratio. These effects were significantly inhibited by glutamine. Treatment with the amino acid also resulted in significant decreases of caspase-9, caspase-8 and caspase-3 activities. Double immunofluorescence staining showed co-localization of CHOP and cleaved caspase-3 in colon sections. Phospho-JNK and PARP-1 expression was also significantly higher in TNBS-treated rats, and treatment with glutamine significantly decreased JNK phosphorylation and PARP-1 proteolysis. To directly address the effect of glutamine on ER stress and apoptosis in epithelial cells, the ER stress inducers brefeldin A and tunicamycin were added to Caco-2 cells that were treated with glutamine (5 mM and 10 mM). The significant enhancement in PERK, ATF6 phosphorylated IRE1, BiP and cleaved caspase-3 expression induced by brefeldin A and tunicamycin was partly prevented by glutamine. Data obtained indicated that modulation of ER stress signalling and anti-apoptotic effects contribute to protection by glutamine against damage

  15. Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation

    PubMed Central

    Shanks, Julia; Herring, Neil; Johnson, Errin; Liu, Kun; Li, Dan

    2017-01-01

    Reduced cardiomyocyte excitation–contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague–Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague–Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; P<0.05). In isolated Sprague–Dawley rat stellate neurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; P<0.05), along with increased endoplasmic reticulum and mitochondria Ca2+ load. Similar results were observed in Wistar Kyoto and age-matched spontaneously hypertensive rats, despite no further increase in endoplasmic reticulum load being observed in the spontaneously hypertensive rat (spontaneously hypertensive rats: empty, 0.16±0.04 au, n=18; SERCA: 0.17±0.02 au, n=25). In conclusion, SERCA2a upregulation in cardiac sympathetic neurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and

  16. Förster resonance energy transfer-based sensor targeting endoplasmic reticulum reveals highly oxidative environment

    PubMed Central

    Kolossov, Vladimir L; Leslie, Matthew T; Chatterjee, Abhishek; Sheehan, Bridget M; Kenis, Paul J A; Gaskins, H Rex

    2012-01-01

    The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition. In previous work, we described the FRET-based redox sensor CY-RL7 with a relatively high midpoint redox potential of −143 mV, which is required for monitoring glutathione potentials in the comparatively high oxidative environment of the ER. Here, the efficacy of the CY-RL7 probe was ascertained in the cytosol and ER of live cells with fluorescence microscopy and flow cytometry. The sensor was found to be fully reduced at steady state in the cytosol and became fully oxidized in response to treatment with 1-chloro-2,4-dinitrobenzene, a depletor of reduced glutathione (GSH). In contrast, the probe was strongly oxidized (88%) upon expression in the ER of cultured cells. We also examined the responsiveness of the ER sensor to perturbations in cellular glutathione homeostasis. We observed that the reductive level of the FRET sensor was increased two-fold to about 28% in cells pretreated with N-acetylcysteine, a substrate for GSH synthesis. Finally, we evaluated the responsiveness of CY-RL7 and roGFP1-iL to various perturbations of cellular glutathione homeostasis to address the divergence in the specificity of these two probes. Together, the present data generated with genetically encoded green fluorescent protein (GFP)-based glutathione probes highlight the complexity of the ER redox environment and indicate that the ER

  17. Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, h...

  18. Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release

    PubMed Central

    Kim, Jae Hong; Johannes, Ludger; Goud, Bruno; Antony, Claude; Lingwood, Clifford A.; Daneman, Richard; Grinstein, Sergio

    1998-01-01

    The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by [H+]-sensitive probes. We devised a procedure for the dynamic, noninvasive measurement of pH in the lumen of the endoplasmic reticulum in intact mammalian cells. A recombinant form of the B subunit of Shiga toxin, previously modified to include a carboxyl-terminal KDEL sequence and a pH-sensitive fluorophore, was used for a two-stage delivery strategy. Retrograde traffic of endogenous lipids was harnessed to target this protein to the Golgi complex, followed by retrieval to the endoplasmic reticulum (ER) by KDEL receptors. Immunofluorescence and immunoelectron microscopy were used to verify the subcellular localization of the modified B fragment. Fluorescence ratio imaging and two independent calibration procedures were applied to determine the pH of the ER in situ. We found that the pH of the endoplasmic reticulum is near neutral and is unaffected during agonist-induced release of calcium. The ER was found to be highly permeable to H+ (equivalents), so that the prevailing [H+] is susceptible to alterations in the cytosolic pH. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplasmic reticulum pH. PMID:9501204

  19. Cis-element of the rice PDIL2-3 promoter is responsible for inducing the endoplasmic reticulum stress response.

    PubMed

    Takahashi, Hideyuki; Wang, Shuyi; Hayashi, Shimpei; Wakasa, Yuhya; Takaiwa, Fumio

    2014-05-01

    A protein disulfide isomerase (PDI) family oxidoreductase, PDIL2-3, is involved in endoplasmic reticulum (ER) stress responses in rice. We identified a critical cis-element required for induction of the ER stress response. The activation of PDIL2-3 in response to ER stress strongly depends on the IRE1-OsbZIP50 signaling pathway.

  20. Temperature- and acceptor-specificity of cell-free vesicular transfer from transitional endoplasmic reticulum to the cis Golgi apparatus.

    PubMed Central

    Dunkle, S; Reust, T; Nowack, D D; Waits, L; Paulik, M; Morre, D M; Morre, D J

    1992-01-01

    The temperature dependence and specificity of transfer of membrane constituents from donor transitional endoplasmic reticulum to the cis Golgi apparatus were investigated using a cell-free system from rat liver. The radiolabelled transitional endoplasmic reticulum donors were prepared from slices of rat liver prelabelled with [14C]leucine. The acceptor Golgi apparatus elements were unlabelled and immobilized on nitrocellulose. When Golgi apparatus stacks were separated by preparative free-flow electrophoresis into subfractions enriched in cisternae derived from the cis, medial and trans portions of the stack respectively, efficient specific transfer was observed only to cis elements. Trans elements were devoid of specific acceptor capacity. Similarly, when transfer was determined as a function of temperature, a transition was observed in transfer activity between 12 degrees C and 18 degrees C similar to that seen in vivo for formation of the so-called 16 degrees C cis Golgi-located membrane compartment. Transfer at temperatures below 16 degrees C and transfer to trans Golgi apparatus compartments at temperatures either above or below 16 degrees C was similar and unspecific. The unspecific transfer at low temperature was pH independent, whereas specific transfer was greatest at the physiological pH of 7, and was reduced to 10% and 18% of that occurring at pH 8 and pH 5.5 respectively. These findings show that the cell-free system derived from rat liver exhibits a high degree of fidelity to transfer in vivo, an efficiency approaching that observed in vivo, and a nearly absolute acceptor specificity for cis Golgi apparatus. The acceptor-, temperature- and pH-specificity of the cell-free transfer, as well as the saturation kinetics exhibited with respect to acceptor Golgi apparatus, support the concept of transition-vesicle-specific docking sites of finite number associated with cis Golgi apparatus cisternae. Images Fig. 4. PMID:1472010

  1. Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors

    SciTech Connect

    Baumann, O.; Walz, B. ); Somlyo, A.V.; Somlyo, A.P. )

    1991-02-01

    Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental compositon of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 {plus minus} 1.1 mmol/kg (dry weight). During a 3-sec nonsaturating light stimulus, {approximately}50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was {approximately}0.7. Our results show unambiguously that the ER is the source of Ca{sup 2+} release during cell stimulation and suggest the Mg{sup 2+} can nearly balance the charge movement of Ca{sup 2+}.

  2. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  3. Endoplasmic reticulum stress, a new wrestler, in the pathogenesis of idiopathic pulmonary fibrosis

    PubMed Central

    Zhang, Lei; Wang, Yi; Pandupuspitasari, Nuruliarizki Shinta; Wu, Guorao; Xiang, Xudong; Gong, Quan; Xiong, Weining; Wang, Cong-Yi; Yang, Ping; Ren, Boxu

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) has attracted extensive attention for its unexplained progressive lung scarring, short median survival and its unresponsiveness to traditional therapies. Despite extensive studies, the mechanisms underlying IPF pathoetiologies, however, remain poorly understood. Recent advances delineated a potential function of endoplasmic reticulum (ER) stress in meeting the need of fibrotic response, which pinpointed a critical role for the unfolded protein response (UPR) pathways in IPF pathogenesis. In this review, we highlight the effect of ER stress and the activation of UPR on the survival, differentiation, function and proliferation of major profibrotic cells in lung tissues during the course of IPF, and discuss the feasibility whether targeting UPR components could be an orientation for developing effective therapeutic strategies against this devastating disorder in clinical settings. PMID:28337301

  4. Targeting the hallmarks of cancer with therapy-induced endoplasmic reticulum (ER) stress

    PubMed Central

    Garg, Abhishek D; Maes, Hannelore; van Vliet, Alexander R; Agostinis, Patrizia

    2015-01-01

    The endoplasmic reticulum (ER) is at the center of a number of vital cellular processes such as cell growth, death, and differentiation, crosstalk with immune or stromal cells, and maintenance of proteostasis or homeostasis, and ER functions have implications for various pathologies including cancer. Recently, a number of major hallmarks of cancer have been delineated that are expected to facilitate the development of anticancer therapies. However, therapeutic induction of ER stress as a strategy to broadly target multiple hallmarks of cancer has been seldom discussed despite the fact that several primary or secondary ER stress-inducing therapies have been found to exhibit positive clinical activity in cancer patients. In the present review we provide a brief historical overview of the major discoveries and milestones in the field of ER stress biology with important implications for anticancer therapy. Furthermore, we comprehensively discuss possible strategies enabling the targeting of multiple hallmarks of cancer with therapy-induced ER stress. PMID:27308392

  5. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress

    PubMed Central

    Yang, Rui; Teng, Xu; Li, Hui; Xue, Hong-Mei; Guo, Qi; Xiao, Lin; Wu, Yu-Ming

    2016-01-01

    In this study, the vitamin D3 plus nicotine (VDN) model of rats was used to prove that H2S alleviates vascular calcification (VC) and phenotype transformation of vascular smooth muscle cells (VSMC). Besides, H2S can also inhibit endoplasmic reticulum stress (ERS) of calcified aortic tissues. The effect of H2S on alleviating VC and phenotype transformation of VSMC can be blocked by TM, while PBA also alleviated VC and phenotype transformation of VSMC that was similar to the effect of H2S. These results suggest that H2S may alleviate rat aorta VC by inhibiting ERS, providing new target and perspective for prevention and treatment of VC. PMID:27022436

  6. Dysfunctional tubular endoplasmic reticulum constitutes a pathological feature of Alzheimer's disease.

    PubMed

    Sharoar, M G; Shi, Q; Ge, Y; He, W; Hu, X; Perry, G; Zhu, X; Yan, R

    2016-09-01

    Pathological features in Alzheimer's brains include mitochondrial dysfunction and dystrophic neurites (DNs) in areas surrounding amyloid plaques. Using a mouse model that overexpresses reticulon 3 (RTN3) and spontaneously develops age-dependent hippocampal DNs, here we report that DNs contain both RTN3 and REEPs, topologically similar proteins that can shape tubular endoplasmic reticulum (ER). Importantly, ultrastructural examinations of such DNs revealed gradual accumulation of tubular ER in axonal termini, and such abnormal tubular ER inclusion is found in areas surrounding amyloid plaques in biopsy samples from Alzheimer's disease (AD) brains. Functionally, abnormally clustered tubular ER induces enhanced mitochondrial fission in the early stages of DN formation and eventual mitochondrial degeneration at later stages. Furthermore, such DNs are abrogated when RTN3 is ablated in aging and AD mouse models. Hence, abnormally clustered tubular ER can be pathogenic in brain regions: disrupting mitochondrial integrity, inducing DNs formation and impairing cognitive function in AD and aging brains.

  7. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    PubMed Central

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  8. Targeting the hallmarks of cancer with therapy-induced endoplasmic reticulum (ER) stress.

    PubMed

    Garg, Abhishek D; Maes, Hannelore; van Vliet, Alexander R; Agostinis, Patrizia

    2015-01-01

    The endoplasmic reticulum (ER) is at the center of a number of vital cellular processes such as cell growth, death, and differentiation, crosstalk with immune or stromal cells, and maintenance of proteostasis or homeostasis, and ER functions have implications for various pathologies including cancer. Recently, a number of major hallmarks of cancer have been delineated that are expected to facilitate the development of anticancer therapies. However, therapeutic induction of ER stress as a strategy to broadly target multiple hallmarks of cancer has been seldom discussed despite the fact that several primary or secondary ER stress-inducing therapies have been found to exhibit positive clinical activity in cancer patients. In the present review we provide a brief historical overview of the major discoveries and milestones in the field of ER stress biology with important implications for anticancer therapy. Furthermore, we comprehensively discuss possible strategies enabling the targeting of multiple hallmarks of cancer with therapy-induced ER stress.

  9. Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

    PubMed Central

    Romero-Brey, Inés; Bartenschlager, Ralf

    2016-01-01

    The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

  10. Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis

    PubMed Central

    Boenisch, Marike Johanne; Broz, Karen Lisa; Purvine, Samuel Owen; Chrisler, William Byron; Nicora, Carrie Diana; Connolly, Lanelle Reine; Freitag, Michael; Baker, Scott Edward; Kistler, Harold Corby

    2017-01-01

    Compartmentalization of metabolic pathways to particular organelles is a hallmark of eukaryotic cells. Knowledge of the development of organelles and attendant pathways under different metabolic states has been advanced by live cell imaging and organelle specific analysis. Nevertheless, relatively few studies have addressed the cellular localization of pathways for synthesis of fungal secondary metabolites, despite their importance as bioactive compounds with significance to medicine and agriculture. When triggered to produce sesquiterpene (trichothecene) mycotoxins, the endoplasmic reticulum (ER) of the phytopathogenic fungus Fusarium graminearum is reorganized both in vitro and in planta. Trichothecene biosynthetic enzymes accumulate in organized smooth ER with pronounced expansion at perinuclear- and peripheral positions. Fluorescence tagged trichothecene biosynthetic proteins co-localize with the modified ER as confirmed by co-fluorescence and co-purification with known ER proteins. We hypothesize that changes to the fungal ER represent a conserved process in specialized eukaryotic cells such as in mammalian hepatocytes and B-cells. PMID:28287158

  11. Endothelial Dysfunction in Diabetes Mellitus: Possible Involvement of Endoplasmic Reticulum Stress?

    PubMed Central

    Basha, Basma; Samuel, Samson Mathews; Triggle, Chris R.; Ding, Hong

    2012-01-01

    The vascular complications of diabetes mellitus impose a huge burden on the management of this disease. The higher incidence of cardiovascular complications and the unfavorable prognosis among diabetic individuals who develop such complications have been correlated to the hyperglycemia-induced oxidative stress and associated endothelial dysfunction. Although antioxidants may be considered as effective therapeutic agents to relieve oxidative stress and protect the endothelium, recent clinical trials involving these agents have shown limited therapeutic efficacy in this regard. In the recent past experimental evidence suggest that endoplasmic reticulum (ER) stress in the endothelial cells might be an important contributor to diabetes-related vascular complications. The current paper contemplates the possibility of the involvement of ER stress in endothelial dysfunction and diabetes-associated vascular complications. PMID:22474423

  12. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib.

    PubMed

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy.

  13. Endoplasmic Reticulum Stress Signaling in Mammalian Oocytes and Embryos: Life in the Balance

    PubMed Central

    Latham, Keith E.

    2015-01-01

    Mammalian oocytes and embryos are exquisitely sensitive to a wide range of insults related to physical stress, chemical exposure, and exposures to adverse maternal nutrition or health status. Although cells manifest specific responses to various stressors, many of these stressors intersect at the endoplasmic reticulum, where disruptions in protein folding and production of reactive oxygen species initiate downstream signaling events. These signals modulate mRNA translation and gene transcription, leading to recovery, activation of autophagy, or with severe and prolonged stress, apoptosis. ER stress signaling has recently come to the fore as a major contributor to embryo demise. Accordingly, agents that modulate or inhibit ER stress signaling have yielded beneficial effects on embryo survival and long-term developmental potential. We review here the mechanisms of ER stress signaling, their connections to mammalian oocytes and embryos, and the promising indications that interventions in this pathway may provide new opportunities for improving mammalian reproduction and health. PMID:25805126

  14. The roles of endoplasmic reticulum stress response in female mammalian reproduction.

    PubMed

    Yang, Yanzhou; Pei, Xiuying; Jin, Yaping; Wang, Yanrong; Zhang, Cheng

    2016-03-01

    Endoplasmic reticulum stress (ERS) activates a protective pathway, called the unfold protein response, for maintaining cellular homeostasis, but cellular apoptosis is triggered by excessive or persistent ERS. Several recent studies imply that the ERS response might have broader physiological roles in the various reproductive processes of female mammals, including embryo implantation, decidualization, preimplantation embryonic development, follicle atresia, and the development of the placenta. This review summarizes the existing data concerning the molecular and biological roles of the ERS response. The study of the functions of the ERS response in mammalian reproduction might provide novel insights into and an understanding of reproductive cell survival and apoptosis under physiological and pathological conditions. The ERS response is a novel signaling pathway for reproductive cell survival and apoptosis. Infertility might be a result of disturbing the ERS response during the process of female reproduction.

  15. Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

    PubMed Central

    Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

    2016-01-01

    Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

  16. Synthesis of Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Living Mammalian Cells**

    PubMed Central

    Matthew Meinig, J.; Fu, Liqiang; Peterson, Blake R.

    2015-01-01

    The endoplasmic reticulum (ER) plays critical roles in the processing of secreted and transmembrane proteins. To deliver small molecules to this organelle, we synthesized fluorinated hydrophobic analogues of the fluorophore rhodol. These cell-permeable fluorophores are exceptionally bright, with quantum yields of ~ 0.8, and specifically accumulate in the ER of living HeLa cells, as imaged by confocal laser scanning microscopy. To target a biological pathway controlled by the ER, we linked a fluorinated hydrophobic rhodol to 5-nitrofuran-2-acrylaldehyde. In contrast to an untargeted nitrofuran warhead, delivery of this electrophilic nitrofuran to the ER by the rhodol resulted in cytotoxicity comparable to the ER-targeted cytotoxin eeyarestatin I, and specifically inhibited protein processing by the ubiquitin-proteasome system. Fluorinated hydrophobic rhodols represent outstanding fluorophores that enable delivery of small molecules for targeting of ER-associated proteins and pathways. PMID:26118368

  17. High osmotic pressure increases reactive oxygen species generation in rabbit corneal epithelial cells by endoplasmic reticulum

    PubMed Central

    Wang, Peng; Sheng, Minjie; Li, Bing; Jiang, Yaping; Chen, Yihui

    2016-01-01

    Tear high osmotic pressure (HOP) has been recognized as the core mechanism underlying ocular surface inflammation, injury and symptoms and is closely associated with many ocular surface diseases, especially dry eye. The endoplasmic reticulum (ER) is a multi-functional organelle responsible for protein synthesis, folding and transport, biological synthesis of lipids, vesicle transport and intracellular calcium storage. Accumulation of unfolded proteins and imbalance of calcium ion in the ER would induce ER stress and protective unfolded protein response (UPR). Many studies have demonstrated that ER stress can induce cell apoptosis. However, the association between tear HOP and ER stress has not been studied systematically. In the present study, rabbit corneal epithelial cells were treated with HOP and results showed that the production of reactive oxygen species increased markedly, which further activated the ER signaling pathway and ultimately induced cell apoptosis. These findings shed new lights on the pathogenesis and clinical treatment of dry eye and other ocular surface diseases. PMID:27158374

  18. Endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation.

    PubMed

    Meyerovich, Kira; Ortis, Fernanda; Allagnat, Florent; Cardozo, Alessandra K

    2016-07-01

    Insulin-secreting pancreatic β-cells are extremely dependent on their endoplasmic reticulum (ER) to cope with the oscillatory requirement of secreted insulin to maintain normoglycemia. Insulin translation and folding rely greatly on the unfolded protein response (UPR), an array of three main signaling pathways designed to maintain ER homeostasis and limit ER stress. However, prolonged or excessive UPR activation triggers alternative molecular pathways that can lead to β-cell dysfunction and apoptosis. An increasing number of studies suggest a role of these pro-apoptotic UPR pathways in the downfall of β-cells observed in diabetic patients. Particularly, the past few years highlighted a cross talk between the UPR and inflammation in the context of both type 1 (T1D) and type 2 diabetes (T2D). In this article, we describe the recent advances in research regarding the interplay between ER stress, the UPR, and inflammation in the context of β-cell apoptosis leading to diabetes.

  19. Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections

    PubMed Central

    Lim, Yun-Ji; Yi, Min-Hee; Choi, Ji-Ae; Lee, Junghwan; Han, Ji-Ye; Jo, Sung-Hee; Oh, Sung-Man; Cho, Hyun Jin; Kim, Dong Woon; Kang, Min-Woong; Song, Chang-Hwa

    2016-01-01

    Alteration of macrophage function has an important regulatory impact on the survival of intracellular mycobacteria. We found that macrophages infected with attenuated Mycobacterium tuberculosis (Mtb) strain H37Ra had elevated expression of M1-related molecules, whereas the M2 phenotype was dominant in macrophages infected with virulent Mtb H37Rv. Further, the TLR signalling pathway played an important role in modulating macrophage polarization against Mtb infection. Interestingly, endoplasmic reticulum (ER) stress was significantly increased in M1 polarized macrophages and these macrophages effectively removed intracellular Mtb, indicating that ER stress may be an important component of the host immune response to Mtb in M1 macrophages. This improved understanding of the mechanisms that regulate macrophage polarization could provide new therapeutic strategies for tuberculosis. PMID:27845414

  20. STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites.

    PubMed

    Wilhelm, Léa P; Wendling, Corinne; Védie, Benoît; Kobayashi, Toshihide; Chenard, Marie-Pierre; Tomasetto, Catherine; Drin, Guillaume; Alpy, Fabien

    2017-04-04

    StAR-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that creates endoplasmic reticulum (ER)-endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill-defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3-mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER-endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER-anchored partner, Vesicle-associated membrane protein-associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER-endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.

  1. Mechanisms of alcohol-induced endoplasmic reticulum stress and organ injuries.

    PubMed

    Ji, Cheng

    2012-01-01

    Alcohol is readily distributed throughout the body in the blood stream and crosses biological membranes, which affect virtually all biological processes inside the cell. Excessive alcohol consumption induces numerous pathological stress responses, part of which is endoplasmic reticulum (ER) stress response. ER stress, a condition under which unfolded/misfolded protein accumulates in the ER, contributes to alcoholic disorders of major organs such as liver, pancreas, heart, and brain. Potential mechanisms that trigger the alcoholic ER stress response are directly or indirectly related to alcohol metabolism, which includes toxic acetaldehyde and homocysteine, oxidative stress, perturbations of calcium or iron homeostasis, alterations of S-adenosylmethionine to S-adenosylhomocysteine ratio, and abnormal epigenetic modifications. Interruption of the ER stress triggers is anticipated to have therapeutic benefits for alcoholic disorders.

  2. Calcium release from intra-axonal endoplasmic reticulum leads to axon degeneration through mitochondrial dysfunction.

    PubMed

    Villegas, Rosario; Martinez, Nicolas W; Lillo, Jorge; Pihan, Phillipe; Hernandez, Diego; Twiss, Jeffery L; Court, Felipe A

    2014-05-21

    Axonal degeneration represents an early pathological event in neurodegeneration, constituting an important target for neuroprotection. Regardless of the initial injury, which could be toxic, mechanical, metabolic, or genetic, degeneration of axons shares a common mechanism involving mitochondrial dysfunction and production of reactive oxygen species. Critical steps in this degenerative process are still unknown. Here we show that calcium release from the axonal endoplasmic reticulum (ER) through ryanodine and IP3 channels activates the mitochondrial permeability transition pore and contributes to axonal degeneration triggered by both mechanical and toxic insults in ex vivo and in vitro mouse and rat model systems. These data reveal a critical and early ER-dependent step during axonal degeneration, providing novel targets for axonal protection in neurodegenerative conditions.

  3. Involvement of VAT-1 in Phosphatidylserine Transfer from the Endoplasmic Reticulum to Mitochondria.

    PubMed

    Junker, Mirco; Rapoport, Tom A

    2015-12-01

    Mitochondria receive phosphatidylserine (PS) from the endoplasmic reticulum (ER), but how PS is moved from the ER to mitochondria is unclear. Current models postulate a physical link between the organelles, but no involvement of cytosolic proteins. Here, we have reconstituted PS transport from the ER to mitochondria in vitro using Xenopus egg components. Transport is independent of ER proteins, but is dependent on a cytosolic factor that has a preferential affinity for PS. Crosslinking with a photoactivatable PS analog identified VAT-1 as a candidate for a cytosolic PS transport protein. Recombinant, purified VAT-1 stimulated PS transport into mitochondria and depletion of VAT-1 from Xenopus cytosol with specific antibodies led to a reduction of transport. Our results suggest that cytosolic factors have a role in PS transport from the ER to mitochondria, implicate VAT-1 in the transport process, and indicate that physical contact between the organelles is not essential.

  4. Modulation of calcium signalling by the endoplasmic reticulum in Carassius neurons.

    PubMed

    Lukyanets, Igor A; Lukyanetz, Elena A

    2013-04-19

    It is known that endoplasmic reticulum (ER), being a calcium store participates in the regulation of intracellular calcium concentration. Ca-ATPase of the ER is one of the crucial agents providing the calcium-accumulating function of this intracellular structure. We studied the role of the ER in modulation of calcium signalling in Carassius neurons using a Ca2+-imaging technique. We tested the role of the ER in the maintenance of a steady state calcium level in the cytoplasm and in modulation of Ca2+ transients evoked by cell depolarizations. The ER calcium stores were depleted using inhibitors of ER Ca-ATPase, which provided blocking of Ca2+ uptake by the ER. Our experiments firstly showed that the ER can significantly modulate the characteristics of intracellular calcium signals in Carassius neurons during their activity. These findings also indicate that the ER modulates the shape of Ca2+ signals rather than the basal level of intracellular Ca2+ in these neurons.

  5. Endoplasmic reticulum-associated protein quality control and degradation: genome-wide screen for ERAD components.

    PubMed

    Schäfer, Antje; Wolf, Dieter H

    2005-01-01

    In this chapter, a genetic approach is presented that leads to the isolation of mutants and to the identification of proteins involved in protein quality control and endoplasmic reticulum-associated degradation (ERAD). The method makes use of a genomic screen of a yeast deletion library (EUROSCARF). Transformation of each of the approx 5000 strains deleted in one nonvital gene each with a CPY* chimera containing CPY* C-terminally fused to a transmembrane domain and the cytosolic Leu2 protein (3-isopropylmalate dehydrogenase) constitutes the basic screening procedure. Because of a Leu2p deficiency in all deletion strains, cells can grow only when the CTL* chimera is present. As the CPY* module of CTL* will be recognized in ERAD-proficient cells, CTL* will be degraded and the strain is unable to grow. Therefore the absence of genes necessary for ER quality control and ERAD will allow cell growth and indicate the necessity of the respective gene for these processes.

  6. Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10.

    PubMed

    Ellenrieder, Lars; Opaliński, Łukasz; Becker, Lars; Krüger, Vivien; Mirus, Oliver; Straub, Sebastian P; Ebell, Katharina; Flinner, Nadine; Stiller, Sebastian B; Guiard, Bernard; Meisinger, Chris; Wiedemann, Nils; Schleiff, Enrico; Wagner, Richard; Pfanner, Nikolaus; Becker, Thomas

    2016-10-10

    The endoplasmic reticulum-mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.

  7. Endoplasmic Reticulum Glucosidase II Is Required for Pathogenicity of Ustilago maydisW⃞

    PubMed Central

    Schirawski, Jan; Böhnert, Heidi U.; Steinberg, Gero; Snetselaar, Karen; Adamikowa, Lubica; Kahmann, Regine

    2005-01-01

    We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic α-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant–fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response. PMID:16272431

  8. Elevated mitochondria-coupled NAD(P)H in endoplasmic reticulum of dopamine neurons

    PubMed Central

    Tucker, Kristal R.; Cavolo, Samantha L.; Levitan, Edwin S.

    2016-01-01

    Pyridine nucleotides are redox coenzymes that are critical in bioenergetics, metabolism, and neurodegeneration. Here we use brain slice multiphoton microscopy to show that substantia nigra dopamine neurons, which are sensitive to stress in mitochondria and the endoplasmic reticulum (ER), display elevated combined NADH and NADPH (i.e., NAD(P)H) autofluorescence. Despite limited mitochondrial mass, organellar NAD(P)H is extensive because much of the signal is derived from the ER. Remarkably, even though pyridine nucleotides cannot cross mitochondrial and ER membranes, inhibiting mitochondrial function with an uncoupler or interrupting the electron transport chain with cyanide (CN−) alters ER NAD(P)H. The ER CN− response can occur without a change in nuclear NAD(P)H, raising the possibility of redox shuttling via the cytoplasm locally between neuronal mitochondria and the ER. We propose that coregulation of NAD(P)H in dopamine neuron mitochondria and ER coordinates cell redox stress signaling by the two organelles. PMID:27582392

  9. Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Leitman, Julia; Ulrich Hartl, F.; Lederkremer, Gerardo Z.

    2013-11-01

    In Huntington’s disease, as in other neurodegenerative diseases, it was initially thought that insoluble protein aggregates are the toxic species. However, growing evidence implicates soluble oligomeric polyglutamine-expanded huntingtin in cytotoxicity. Here we show that pathogenic huntingtin inhibits endoplasmic reticulum (ER)-associated degradation and induces ER stress before its aggregation into visible inclusions. All three branches of the unfolded protein response are activated. ER stress can be compensated by overexpression of p97/VCP, suggesting its sequestration by pathogenic huntingtin as a main cause. Stress correlates with the presence of huntingtin oligomers and is independent of continual huntingtin synthesis. Stress levels, measured in striatal neurons, are stabilized but only slowly subside on huntingtin aggregation into inclusions. Our results can be explained by the constant conversion of huntingtin monomers to toxic oligomers; large aggregates sequester the former, precluding further conversion, whereas pre-existing toxic oligomers are only gradually depleted.

  10. Endothelial dysfunction in diabetes mellitus: possible involvement of endoplasmic reticulum stress?

    PubMed

    Basha, Basma; Samuel, Samson Mathews; Triggle, Chris R; Ding, Hong

    2012-01-01

    The vascular complications of diabetes mellitus impose a huge burden on the management of this disease. The higher incidence of cardiovascular complications and the unfavorable prognosis among diabetic individuals who develop such complications have been correlated to the hyperglycemia-induced oxidative stress and associated endothelial dysfunction. Although antioxidants may be considered as effective therapeutic agents to relieve oxidative stress and protect the endothelium, recent clinical trials involving these agents have shown limited therapeutic efficacy in this regard. In the recent past experimental evidence suggest that endoplasmic reticulum (ER) stress in the endothelial cells might be an important contributor to diabetes-related vascular complications. The current paper contemplates the possibility of the involvement of ER stress in endothelial dysfunction and diabetes-associated vascular complications.

  11. Endoplasmic reticulum stress and its effects on renal tubular cells apoptosis in ischemic acute kidney injury.

    PubMed

    Xu, Yan; Guo, Min; Jiang, Wei; Dong, Hui; Han, Yafei; An, Xiao-Fei; Zhang, Jisheng

    2016-06-01

    Ischemia is the most frequent cause of acute kidney injury (AKI), which is characterized by apoptosis of renal tubular cell. A common result of ischemia in AKI is dysfunction of endoplasmic reticulum (ER), which causes the protein-folding capacity to lag behind the protein-folding load. The abundance of misfolded proteins stressed the ER and results in induction of the unfolded protein response (UPR). While the UPR is an adaptive response, over time it can result in apoptosis when cells are unable to recover quickly. Recent research suggests that ER stress is a major factor in renal tubular cell apoptosis resulting from ischemic AKI. Thus, ER stress may be an important new progression factor in the pathology of ischemic AKI. In this article, we review UPR signaling, describe pathology and pathophysiology mechanisms of ischemic AKI, and highlight the dual function of ER stress on renal tubular cell apoptosis.

  12. [Calcium transport in endoplasmic reticulum of the rat liver during lipid peroxidation].

    PubMed

    Gubskiĭ, Iu I; Kurskiĭ, M D; Zadorina, O V; Fedorov, A N; Briuzgina, T S; Iurzhenko, N N

    1990-01-01

    Some parameters of calcium transport in rat liver microsomes under conditions of lipoperoxidation activation modelled by antioxidant deficiency (AOD) were studied. This process was shown to be associated with a sharp stimulation of NADPH- and ascorbate-dependent lipid peroxidation in hepatocyte endoplasmic reticulum. The activation of lipid peroxidation was accompanied by disturbances in the kinetic properties of Ca2(+)-ATPase. This was paralleled with a considerable decrease of the ATP-dependent 45Ca-accumulation, increase in the passive permeability of microsomal vesicles for Ca2+ and Ca2+ elevation in the microsomal fraction. The AOD-induced diminution of the Ca2(+)-pump efficiency was slightly prevented by injections of rats with the antioxidants, alpha-tocopherol acetate and ionol which enable Ca2+ compartmentation correction in liver cytosol and membrane fractions.

  13. Endoplasmic reticulum stress as a primary pathogenic mechanism leading to age-related macular degeneration.

    PubMed

    Libby, Richard T; Gould, Douglas B

    2010-01-01

    Age-related macular degeneration (AMD) is a multi-factorial disease and a leading cause of blindness. Proteomic and genetic data suggest that activation or de-repression of the alternate complement cascade of innate immunity is involved in end-stage disease. Several lines of evidence suggest that production of reactive oxygen species and chronic oxidative stress lead to protein and lipid modifications that initiate the complement cascade. Understanding the triggers of these pathogenic pathways and the site of the primary insult will be important for development of targeted therapeutics. Endoplasmic reticulum (ER) stress from misfolded mutant proteins and other sources are an important potential tributary mechanism. We propose that misfolded-protein-induced ER stress in the retinal-pigmented epithelium and/or choroid could lead to chronic oxidative stress, complement deregulation and AMD. Small molecules targeted to ER stress and oxidative stress could allow for a shift from disease treatment to disease prevention.

  14. Salubrinal Alleviates Pressure Overload-Induced Cardiac Hypertrophy by Inhibiting Endoplasmic Reticulum Stress Pathway

    PubMed Central

    Rani, Shilpa; Sreenivasaiah, Pradeep Kumar; Cho, Chunghee; Kim, Do Han

    2017-01-01

    Pathological hypertrophy of the heart is closely associated with endoplasmic reticulum stress (ERS), leading to maladaptations such as myocardial fibrosis, induction of apoptosis, and cardiac dysfunctions. Salubrinal is a known selective inhibitor of protein phosphatase 1 (PP1) complex involving dephosphorylation of phospho-eukaryotic translation initiation factor 2 subunit (p-eIF2)-α, the key signaling process in the ERS pathway. In this study, the effects of salubrinal were examined on cardiac hypertrophy using the mouse model of transverse aortic constriction (TAC) and cell model of neonatal rat ventricular myocytes (NRVMs). Treatment of TAC-induced mice with salubrinal (0.5 mg·kg−1·day−1) alleviated cardiac hypertrophy and tissue fibrosis. Salubrinal also alleviated hypertrophic growth in endothelin 1 (ET1)-treated NRVMs. Therefore, the present results suggest that salubrinal may be a potentially efficacious drug for treating pathological cardiac remodeling. PMID:28152298

  15. Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis.

    PubMed

    Schirawski, Jan; Böhnert, Heidi U; Steinberg, Gero; Snetselaar, Karen; Adamikowa, Lubica; Kahmann, Regine

    2005-12-01

    We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic alpha-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.

  16. Coordination of stress, Ca2+, and immunogenic signaling pathways by PERK at the endoplasmic reticulum.

    PubMed

    van Vliet, Alexander R; Garg, Abhishek D; Agostinis, Patrizia

    2016-07-01

    The endoplasmic reticulum (ER) is the main coordinator of intracellular Ca2+ signaling, protein synthesis, and folding. The ER is also implicated in the formation of contact sites with other organelles and structures, including mitochondria, plasma membrane (PM), and endosomes, thereby orchestrating through interorganelle signaling pathways, a variety of cellular responses including Ca2+ homeostasis, metabolism, and cell death signaling. Upon loss of its folding capacity, incited by a number of stress signals including those elicited by various anticancer therapies, the unfolded protein response (UPR) is launched to restore ER homeostasis. The ER stress sensor protein kinase RNA-like ER kinase (PERK) is a key mediator of the UPR and its role during ER stress has been largely recognized. However, growing evidence suggests that PERK may govern signaling pathways through UPR-independent functions. Here, we discuss emerging noncanonical roles of PERK with particular relevance for the induction of danger or immunogenic signaling and interorganelle communication.

  17. ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from the Endoplasmic Reticulum of Leek Cells

    PubMed Central

    Sturbois-Balcerzak, Bénédicte; Vincent, Patrick; Maneta-Peyret, Lilly; Duvert, Michel; Satiat-Jeunemaitre, Béatrice; Cassagne, Claude; Moreau, Patrick

    1999-01-01

    Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625–637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPγ-S prevents vesicle formation. These vesicles correspond to small structures (70–80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells. PMID:10318702

  18. How viruses use the endoplasmic reticulum for entry, replication, and assembly.

    PubMed

    Inoue, Takamasa; Tsai, Billy

    2013-01-01

    To cause infection, a virus enters a host cell, replicates, and assembles, with the resulting new viral progeny typically released into the extracellular environment to initiate a new infection round. Virus entry, replication, and assembly are dynamic and coordinated processes that require precise interactions with host components, often within and surrounding a defined subcellular compartment. Accumulating evidence pinpoints the endoplasmic reticulum (ER) as a crucial organelle supporting viral entry, replication, and assembly. This review focuses on the molecular mechanism by which different viruses co-opt the ER to accomplish these crucial infection steps. Certain bacterial toxins also hijack the ER for entry. An interdisciplinary approach, using rigorous biochemical and cell biological assays coupled with advanced microscopy strategies, will push to the next level our understanding of the virus-ER interaction during infection.

  19. Compartmentalization of the endoplasmic reticulum in the early C. elegans embryos

    PubMed Central

    Lee, Zuo Yen; Prouteau, Manoël

    2016-01-01

    The one-cell Caenorhabditis elegans embryo is polarized to partition fate determinants between the cell lineages generated during its first division. Using fluorescence loss in photobleaching, we find that the endoplasmic reticulum (ER) of the C. elegans embryo is physically continuous throughout the cell, but its membrane is compartmentalized shortly before nuclear envelope breakdown into an anterior and a posterior domain, indicating that a diffusion barrier forms in the ER membrane between these two domains. Using mutants with disorganized ER, we show that ER compartmentalization is independent of the morphological transition that the ER undergoes in mitosis. In contrast, compartmentalization takes place at the position of the future cleavage plane in a par-3–dependent manner. Together, our data indicate that the ER membrane is compartmentalized in cells as diverse as budding yeast, mouse neural stem cells, and the early C. elegans embryo. PMID:27597753

  20. Cell biology of the endoplasmic reticulum and the Golgi apparatus through proteomics.

    PubMed

    Smirle, Jeffrey; Au, Catherine E; Jain, Michael; Dejgaard, Kurt; Nilsson, Tommy; Bergeron, John

    2013-01-01

    Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry.

  1. Brain death is associated with endoplasmic reticulum stress and apoptosis in rat liver.

    PubMed

    Cao, S; Wang, T; Yan, B; Lu, Y; Zhao, Y; Zhang, S

    2014-12-01

    Cell death pathways initiated by stress on the endoplasmic reticulum (ER) have been implicated in a variety of common diseases, such as ischemia/reperfusion injury, diabetes, heart disease, and neurodegenerative disorders. However, the contribution of ER stress to apoptosis and liver injury after brain death is not known. In the present study, we found that brain death induces a variety of signature ER stress markers, including ER stress-specific X box-binding protein 1 and up-regulation of glucose-regulated protein 78. Furthermore, brain death causes up-regulation of C/EBP homologous protein and caspase-12. Consistent with this, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling assay and transmission electron microscopy confirmed apoptosis in the liver after brain death. Taken together, the present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain death-induced apoptosis and liver injury.

  2. Real-time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions

    PubMed Central

    Merksamer, Philip I.; Trusina, Ala; Papa, Feroz R.

    2008-01-01

    SUMMARY Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing “ER stress” whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems. PMID:19026441

  3. Endoplasmic Reticulum and the Unfolded Protein Response: Dynamics and Metabolic Integration

    PubMed Central

    Bravo, Roberto; Parra, Valentina; Gatica, Damián; Rodriguez, Andrea E.; Torrealba, Natalia; Paredes, Felipe; Wang, Zhao V.; Zorzano, Antonio; Hill, Joseph A.; Jaimovich, Enrique; Quest, Andrew F.G.; Lavandero, Sergio

    2013-01-01

    The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of reestablishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies. PMID:23317820

  4. A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

    PubMed Central

    Ravindran, Madhu Sudhan; Bagchi, Parikshit; Inoue, Takamasa; Tsai, Billy

    2015-01-01

    Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event. PMID:26244546

  5. Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

    PubMed Central

    Orsi, Andrea

    2007-01-01

    Prion protein (PrP), the causative agent of transmissible spongiform encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation. PMID:19164910

  6. Tumor Hypoxia Blocks Wnt Processing and Secretion through the Induction of Endoplasmic Reticulum Stress▿

    PubMed Central

    Verras, Meletios; Papandreou, Ioanna; Lim, Ai Lin; Denko, Nicholas C.

    2008-01-01

    Poorly formed tumor blood vessels lead to regions of microenvironmental stress due to depletion of oxygen and glucose and accumulation of waste products (acidosis). These conditions contribute to tumor progression and correlate with poor patient prognosis. Here we show that the microenvironmental stresses found in the solid tumor are able to inhibit the canonical Wnt/β-catenin signaling pathway. However, tumor cells harboring common β-catenin pathway mutations, such as loss of adenomatous polyposis coli, are insensitive to this novel hypoxic effect. The underlying mechanism responsible is hypoxia-induced endoplasmic reticulum (ER) stress that inhibits normal Wnt protein processing and secretion. ER stress causes dissociation between GRP78/BiP and Wnt, an interaction essential for its correct posttranslational processing. Microenvironmental stress can therefore block autocrine and paracrine signaling of the Wnt/β-catenin pathway and negatively affect tumor growth. This study provides a general paradigm relating oxygen status to ER function and growth factor signaling. PMID:18824543

  7. Glucuronide transport across the endoplasmic reticulum membrane is inhibited by epigallocatechin gallate and other green tea polyphenols.

    PubMed

    Révész, Katalin; Tütto, Anna; Margittai, Eva; Bánhegyi, Gábor; Magyar, Judit E; Mandl, József; Csala, Miklós

    2007-01-01

    Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.

  8. Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells

    PubMed Central

    Gafar, Ahmed A.; Draz, Hossam M.; Goldberg, Alexander A.; Bashandy, Mohamed A.; Bakry, Sayed; Khalifa, Mahmoud A.; AbuShair, Walid; Titorenko, Vladimir I.

    2016-01-01

    Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI–LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively

  9. Molecular Species of Phosphatidylinositol-Cycle Intermediates in the Endoplasmic Reticulum and Plasma Membrane†

    PubMed Central

    Shulga, Yulia V.; Myers, David S.; Ivanova, Pavlina T.; Milne, Stephen B.; Brown, H. Alex; Topham, Matthew K.; Epand, Richard M.

    2009-01-01

    Phosphatidylinositol (PI) turnover is a process requiring both the plasma and ER membranes. We have determined the distribution of phosphatidic acid (PA) and PI and their acyl chain compositions in these two subcellular membranes using mass spectrometry. We assessed the role of PI cycling in determining the molecular species and quantity of these lipids by comparing the compositions of the two membranes isolated from embryonic fibroblasts obtained from diacylglycerol kinase epsilon (DGKε) knock out (KO) and wild type (WT) mice. In the KO cells the conversion of arachidonoyl-rich DAG to PA is blocked by the absence of DGKε, resulting in reducing the rate of PI-cycling. The acyl chain composition is very similar for PI or PA in the endoplasmic reticulum (ER) vs. plasma membrane (PM) and for WT vs. KO. However, the acyl chain profile for PI is very different from that for PA. This indicates that DGKε is not facilitating the direct transfer of a specific species of PA between the PM and the ER. About 20% of the PA in the ER membrane has one short acyl chain of 14 carbons or less. These species of PA are not converted into PI but may play a role in stabilizing regions of high positive curvature in the ER. There are also PI species in both the ER and PM for which there is no detectable PA precursor, indicating that these species of PI are unlikely to arise via the PI-cycle. We find that in the PM of KO cells the levels of PI and of PA are decreased about three-fold in comparison with either the PM of WT cells or in comparison with the ER of KO cells. The PI-cycle is slowed in the KO cells, hence the lipid intermediates of the PI-cycle can no longer be interconverted and are depleted from the PI-cycle by conversion to other species. There is less of an effect of the depletion in the ER where de novo synthesis of PA occurs in comparison with the PM. PMID:20000336

  10. Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis

    PubMed Central

    Price, Gareth R; Tauro, Sharyn B; Taupin, Douglas; Thornton, David J; Png, Chin Wen; Crockford, Tanya L; Cornall, Richard J; Adams, Rachel; Kato, Masato; Nelms, Keats A; Hong, Nancy A; Florin, Timothy H. J; Goodnow, Christopher C; McGuckin, Michael A

    2008-01-01

    Background MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. Methods and Findings By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1β, TNF-α, and IFN-γ was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-γ, TNF-α, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar

  11. Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells.

    PubMed

    Gafar, Ahmed A; Draz, Hossam M; Goldberg, Alexander A; Bashandy, Mohamed A; Bakry, Sayed; Khalifa, Mahmoud A; AbuShair, Walid; Titorenko, Vladimir I; Sanderson, J Thomas

    2016-01-01

    Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI-LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively

  12. Involvement of endoplasmic reticulum stress in formalin-induced pain is attenuated by 4-phenylbutyric acid

    PubMed Central

    Zhou, Fan; Zhang, Wei; Zhou, Jianmei; Li, Meirong; Zhong, Feng; Zhang, Yun; Liu, Yuezhu; Wang, Yaping

    2017-01-01

    Background Endoplasmic reticulum (ER) stress is involved in many neurological and inflammatory responses. Peripheral inflammatory responses can induce central sensitization and trigger inflammatory pain. However, there is little research on the relationship between ER stress and inflammatory pain. In this study, we examined whether the ER stress response is involved in peripheral inflammatory pain using a formalin-induced rat pain model. Methods Rats were divided into the following five groups: control, formalin, formalin + vehicle, formalin + 4-phenylbutyric acid (4-PBA) (40 mg/kg) and formalin + 4-PBA (100 mg/kg). Formalin-induced pain was assessed behaviorally by recording licking activity. The expression levels of immunoglobulin-binding protein (BIP), activating transcription factor-6 (ATF6), phosphorylated inositol-requiring enzyme-1 (p-IRE1), phosphorylated protein kinase RNA-like ER kinase (p-PERK) and c-fos were quantitatively assessed by Western blot, and the distribution of BIP, ATF6 and c-fos in the lumbar enlargement of spinal cord were identified by immunohistochemistry in spinal dorsal horn slices. In addition, the concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) in the spinal cord were tested by biochemical measurement and enzyme-linked immunosorbent assay (ELISA), respectively. Results Intraperitoneal injection of 4-PBA at the dose of 100 mg/kg before formalin injection significantly decreased nociceptive behavior in the second phase compared with control, formalin, formalin + vehicle and formalin + 4-PBA (40 mg/kg) (P<0.05). Western blot showed that formalin injection significantly upregulated the expression of BIP, ATF6, p-PERK and c-fos in the spinal cord. This upregulation was reduced by peritoneal injection of 4-PBA (P<0.05), while expression of p-IRE1 was not altered by formalin treatment. Immunohistochemistry revealed markedly increased staining density for BIP, ATF6 and c-fos in the superficial spinal dorsal horn after

  13. α1-antitrypsin Deficiency: A Misfolded Secretory Protein Variant with Unique Effects on the Endoplasmic Reticulum

    PubMed Central

    Perlmutter, David H

    2016-01-01

    In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to accumulation of a misfolded secretory glycoprotein in the endoplasmic reticulum (ER) of liver cells and so ATD has come to be considered a prototypical ER storage disease. It is associated with two major types of clinical disorders, chronic obstructive pulmonary disease (COPD) by loss-of-function mechanisms and hepatic cirrhosis and carcinogenesis by gain-of-function mechanisms. The lung disease predominantly results from proteolytic damage to the pulmonary connective tissue matrix because of reduced levels of protease inhibitor activity of α1-anitrypsin (AT) in the circulating blood and body fluids. Cigarette smoking is a powerful disease-promoting modifier but other modifiers are known to exist because variation in the lung disease phenotype is still found in smoking and non-smoking homozygotes. The liver disease is highly likely to be caused by the proteotoxic effects of intracellular misfolded protein accumulation and a high degree of variation in the hepatic phenotype among affected homozygotes has been hypothetically attributed to genetic and environmental modifiers that alter proteostasis responses. Liver biopsies of homozygotes show intrahepatocytic inclusions with dilation and expansion of the ER and recent studies of iPS-derived hepatocyte-like cells from individuals with ATD indicate that the changes in the ER directly vary with the hepatic phenotype i.e there is much lesser alteration in the ER in cells derived from homozygotes that do not have clinically significant liver disease. From a signaling perspective, studies in mammalian cell line and animal models expressing the classical α1-antitrypsin Z variant (ATZ) have found that ER signaling is perturbed in a relatively unique way with powerful activation of autophagy and the NFκB pathway but relatively limited, if any, UPR signaling. It is still not known how much these unique structural and functional changes and

  14. Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis*

    PubMed Central

    Stocki, Pawel; Chapman, Daniel C.; Beach, Lori A.; Williams, David B.

    2014-01-01

    Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins. PMID:24990953

  15. Tributyltin-induced endoplasmic reticulum stress and its Ca{sup 2+}-mediated mechanism

    SciTech Connect

    Isomura, Midori; Kotake, Yaichiro Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

    2013-10-01

    Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca{sup 2+} signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca{sup 2+} homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca{sup 2+} depletion, and to test this idea, we examined the effect of TBT on intracellular Ca{sup 2+} concentration using fura-2 AM, a Ca{sup 2+} fluorescent probe. TBT increased intracellular Ca{sup 2+} concentration in a TBT-concentration-dependent manner, and Ca{sup 2+} increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca{sup 2+} concentration by releasing Ca{sup 2+} from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca{sup 2+} release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca{sup 2+}.

  16. Excessive training is associated with endoplasmic reticulum stress but not apoptosis in the hypothalamus of mice.

    PubMed

    Pinto, Ana Paula; da Rocha, Alisson Luiz; Pereira, Bruno Cesar; Oliveira, Luciana da Costa; Morais, Gustavo Paroschi; Moura, Leandro Pereira; Ropelle, Eduardo Rochete; Pauli, José Rodrigo; da Silva, Adelino Sanchez Ramos

    2016-12-05

    Downhill running-based overtraining model increases the hypothalamic levels of IL-1β, TNF-α, SOCS3, and pSAPK-JNK. The aim of the present study was to verify the effects of 3 overtraining protocols on the levels of BiP, pIRE-1 (Ser724), pPERK (Thr981), pelF2α (Ser52), ATF-6, GRP-94, caspase 4, caspase 12, pAKT (Ser473), pmTOR (Ser2448), and pAMPK (Thr172) proteins in the mouse hypothalamus. The mice were randomized into the control, overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up), and overtrained by running without inclination (OTR) groups. After the overtraining protocols (i.e., at the end of week 8), hypothalamus was removed and used for immunoblotting. The OTR/down group exhibited increased levels of all of the analyzed endoplasmic reticulum stress markers in the hypothalamus at the end of week 8. The OTR/up and OTR groups exhibited increased levels of BiP, pIRE-1 (Ser724), and pPERK (Thr981) in the hypothalamus at the end of week 8. There were no significant differences in the levels of caspase 4, caspase 12, pAKT (Ser473), pmTOR (Ser2448), and pAMPK (Thr172) between the experimental groups at the end of week 8. In conclusion, the 3 overtraining protocols increased the endoplasmic reticulum stress at the end of week 8.

  17. Rescue of Glaucomatous Neurodegeneration by Differentially Modulating Neuronal Endoplasmic Reticulum Stress Molecules

    PubMed Central

    Yang, Liu; Li, Shaohua; Miao, Linqing; Huang, Haoliang; Liang, Feisi; Teng, Xiuyin; Xu, Lin; Wang, Qizhao; Xiao, Weidong; Ridder, William H.; Ferguson, Toby A.; Chen, Dong Feng; Kaufman, Randal J.

    2016-01-01

    Axon injury is an early event in neurodegenerative diseases that often leads to retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. The connection of these two obviously sequential degenerative events, however, is elusive. Deciphering the upstream signals that trigger the neurodegeneration cascades in both neuronal soma and axon would be a key step toward developing the effective neuroprotectants that are greatly needed in the clinic. We showed previously that optic nerve injury-induced neuronal endoplasmic reticulum (ER) stress plays an important role in retinal ganglion cell (RGC) death. Using two in vivo mouse models of optic neuropathies (traumatic optic nerve injury and glaucoma) and adeno-associated virus–mediated RGC-specific gene targeting, we now show that differential manipulation of unfolded protein response pathways in opposite directions—inhibition of eukaryotic translation initiation factor 2α-C/EBP homologous protein and activation of X-box binding protein 1—promotes both RGC axons and somata survival and preserves visual function. Our results indicate that axon injury-induced neuronal ER stress plays an important role in both axon degeneration and neuron soma death. Neuronal ER stress is therefore a promising therapeutic target for glaucoma and potentially other types of neurodegeneration. SIGNIFICANCE STATEMENT Neuron soma and axon degeneration have distinct molecular mechanisms although they are clearly connected after axon injury. We previously demonstrated that axon injury induces neuronal endoplasmic reticulum (ER) stress and that manipulation of ER stress molecules synergistically promotes neuron cell body survival. Here we investigated the possibility that ER stress also plays a role in axon degeneration and whether ER stress modulation preserves neuronal function in neurodegenerative diseases. Our results suggest that neuronal ER stress is a general mechanism of degeneration for both neuronal

  18. Physiological functions of endoplasmic reticulum stress transducer OASIS in central nervous system.

    PubMed

    Saito, Atsushi

    2014-01-01

    Eukaryotic cells can adapt to endoplasmic reticulum (ER) dysfunction by producing diverse signals from the ER to the cytosol or nucleus. These signaling pathways are collectively known as the unfolded protein response (UPR). The canonical branches of the UPR are mediated by three ER membrane-bound proteins: double-stranded RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6). These ER stress transducers basically play important roles in cell survival after ER stress. Recently, novel types of ER stress transducers that share a region of high sequence similarity with ATF6 have been identified. They have a transmembrane domain, which allows them to associate with the ER, and possess a transcription-activation domain and a basic leucine zipper (bZIP) domain. These membrane-bound bZIP transcription factors include OASIS, BBF2H7 CREBH, CREB4 and Luman, and are collectively referred to as OASIS family members. Despite their structural similarities with ATF6, differences in activating stimuli and tissue distribution indicate specialized functions of each member on regulating UPR signaling in specific organs and tissues. One of them, OASIS, is expressed preferentially in astrocytes in the central nervous system (CNS). OASIS temporally regulates the differentiation from neural precursor cells into astrocytes to promote the expression of Glial Cell Missing 1 through dynamic interactions among OASIS family members followed by accelerating demethylation of the Gfap promoter. This review is a summary of our current understanding of the physiological functions of OASIS in the CNS.

  19. Identification and functional characterization of an endoplasmic reticulum oxidoreductin 1-α gene in Litopenaeus vannamei.

    PubMed

    Zhang, Ze-Zhi; Yuan, Kai; Yue, Hai-Tao; Yuan, Feng-Hua; Bi, Hai-Tao; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-04-01

    In the current study, full-length sequence of endoplasmic reticulum oxidoreductin 1-α (LvERO1-α) was cloned from Litopenaeus vannamei. Real-time RT-PCR results showed that LvERO1-α was highly expressed in hemocytes, gills, and intestines. White spot syndrome virus (WSSV) challenge was performed, and the expression of LvERO1-α and two other downstream genes of the double-stranded RNA-activated protein kinase-like ER kinase-eIF2α (PERK-α) pathway, namely, homocysteine-induced endoplasmic reticulum protein (LvHERP) and acylamino-acid-releasing enzyme (LvAARE), strongly increased in the hemocytes. Flow cytometry assay results indicated that the apoptosis rate of L. vannamei hemocytes in the LvERO1-α knockdown group was significantly lower than that of the controls. Moreover, shrimps with knockdown expression of LvERO1-α exhibited decreased cumulative mortality upon WSSV infection. Downregulation of L. vannamei immunoglobulin-binding protein (LvBip), which had been proven to induce unfolded protein response (UPR) in L. vannamei, did not only upregulate LvERO1-α, LvHERP, and LvAARE in hemocytes, but also increased their apoptosis rate, as well as the shrimp cumulative mortality. Furthermore, reporter gene assay results showed that the promoter of LvERO1-α was activated by L. vannamei activating transcription factor 4, thereby confirming that LvERO1-α was regulated by the PERK-eIF2α pathway. These results suggested that LvERO1-α plays a critical role in WSSV-induced apoptosis, which likely occurs through the WSSV-activated PERK-eIF2α pathway.

  20. Modulation of endoplasmic reticulum stress and cardiomyocyte apoptosis by mulberry leaf diet in experimental autoimmune myocarditis rats

    PubMed Central

    Arumugam, Somasundaram; Thandavarayan, Rajarajan A.; Veeraveedu, Punniyakoti T.; Ma, Meilei; Giridharan, Vijayasree V.; Arozal, Wawaimuli; Sari, Flori R.; Sukumaran, Vijayakumar; Lakshmanan, Arunprasath; Soetikno, Vivian; Suzuki, Kenji; Kodama, Makoto; Watanabe, Kenichi

    2012-01-01

    Mulberry is commonly used as silkworm diet and an alternative medicine in Japan and China, has recently reported to contain many antioxidative flavanoid compounds and having the free radical scavenging effects. Antioxidants reduce cardiac oxidative stress and attenuate cardiac dysfunction in animals with pacing-induced congestive heart failure. Hence we investigated the cardioprotective effect of mulberry leaf powder in rats with experimental autoimmune myocarditis. Eight-week-old Lewis rats immunized with cardiac myosin were fed with either normal chow or a diet containing 5% mulberry leaf powder and were examined on day 21. ML significantly decreased oxidative stress, myocyte apoptosis, cellular infiltration, cardiac fibrosis, mast cell density, myocardial levels of sarco/endo-plasmic reticulum Ca2+ ATPase2, p22phox, receptor for advanced glycation end products, phospho-p38 mitogen activated protein kinase, phospho-c-Jun NH2-terminal protein kinase, glucose regulated protein78, caspase12 and osteopontin levels in EAM rats. These results may suggest that mulberry diet can preserve the cardiac function in experimental autoimmune myocarditis by modulating oxidative stress induced MAPK activation and further afford protection against endoplasmic reticulum stress mediated apoptosis. PMID:22448095

  1. HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

    SciTech Connect

    Luo, Ying; Li, Shu-Jun; Yang, Jian; Qiu, Yuan-Zhen; Chen, Fang-Ping

    2013-09-06

    Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.

  2. Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4

    SciTech Connect

    Brüning, Ansgar Matsingou, Christina; Brem, German Johannes; Rahmeh, Martina; Mylonas, Ioannis

    2012-10-15

    Inhibins and activins are gonadal peptide hormones of the transforming growth factor-β super family with important functions in the reproductive system. By contrast, the recently identified inhibin βE subunit, primarily expressed in liver cells, appears to exert functions unrelated to the reproductive system. Previously shown downregulation of inhibin βE in hepatoma cells and anti-proliferative effects of ectopic inhibin βE overexpression indicated growth-regulatory effects of inhibin βE. We observed a selective re-expression of the inhibin βE subunit in HepG2 hepatoblastoma cells, MCF7 breast cancer cells, and HeLa cervical cancer cells under endoplasmic reticulum stress conditions induced by tunicamycin, thapsigargin, and nelfinavir. Analysis of XPB1 splicing and ATF4 activation revealed that inhibin βE re-expression was associated with induction of the endoplasmic reticulum stress reaction by these drugs. Transfection of an ATF4 expression plasmid specifically induced inhibin βE expression in HeLa cells and indicates inhibin βE as a hitherto unidentified target gene of ATF4, a key transcription factor of the endoplasmic reticulum stress response. Therefore, the inhibin βE subunit defines not only a new player but also a possible new marker for drug-induced endoplasmic reticulum stress. -- Highlights: ► Endoplasmic reticulum stress induces inhibin beta E expression. ► Inhibin beta E is regulated by the transcription factor ATF4. ► Inhibin beta E expression can be used as a marker for drug-induced ER stress.

  3. Astragaloside IV Attenuates Podocyte Apoptosis Mediated by Endoplasmic Reticulum Stress through Upregulating Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 Expression in Diabetic Nephropathy

    PubMed Central

    Guo, Hengjiang; Cao, Aili; Chu, Shuang; Wang, Yi; Zang, Yingjun; Mao, Xiaodong; Wang, Hao; Wang, Yunman; Liu, Cheng; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a central role in the pathogenesis of diabetes. This protein has been recognized as a potential target for diabetic therapy. In this study, we identified astragaloside IV (AS-IV) as a potent modulator of SERCA inhibiting renal injury in diabetic status. Increasing doses of AS-IV (2, 6, and 18 mg kg-1 day-1) were administered intragastrically to db/db mice for 8 weeks. Biochemical and histopathological approaches were conducted to evaluate the therapeutic effects of AS-IV. Cultured mouse podocytes were used to further explore the underlying mechanism in vitro. AS-IV dose-dependently increased SERCA activity and SERCA2 expression, and suppressed ER stress-mediated and mitochondria-mediated apoptosis in db/db mouse kidney. AS-IV also normalized glucose tolerance and insulin sensitivity, improved renal function, and ameliorated glomerulosclerosis and renal inflammation in db/db mice. In palmitate stimulated podocytes, AS-IV markedly improved inhibitions of SERCA activity and SERCA2 expression, restored intracellular Ca2+ homeostasis, and attenuated podocyte apoptosis in a dose-dependent manner with a concomitant abrogation of ER stress as evidenced by the downregulation of GRP78, cleaved ATF6, phospho-IRE1α and phospho-PERK, and the inactivation of both ER stress-mediated and mitochondria-mediated apoptotic pathways. Furthermore, SERCA2b knockdown eliminated the effect of AS-IV on ER stress and ER stress-mediated apoptotic pathway, whereas its overexpression exhibited an anti-apoptotic effect. Our data obtained from in vivo and in vitro studies demonstrate that AS-IV attenuates renal injury in diabetes subsequent to inhibiting ER stress-induced podocyte apoptosis through restoring SERCA activity and SERCA2 expression. PMID:28066247

  4. Endoplasmic Reticulum Oxidative Stress Triggers Tgf-Beta-Dependent Muscle Dysfunction by Accelerating Ascorbic Acid Turnover

    PubMed Central

    Pozzer, Diego; Favellato, Mariagrazia; Bolis, Marco; Invernizzi, Roberto William; Solagna, Francesca; Blaauw, Bert; Zito, Ester

    2017-01-01

    Endoplasmic reticulum (ER) and oxidative stress are two related phenomena that have important metabolic consequences. As many skeletal muscle diseases are triggered by oxidative stress, we explored the chain of events linking a hyperoxidized ER (which causes ER and oxidative stress) with skeletal muscle dysfunction. An unbiased exon expression array showed that the combined genetic modulation of the two master ER redox proteins, selenoprotein N (SEPN1) and endoplasmic oxidoreductin 1 (ERO1), led to an SEPN1-related myopathic phenotype due to excessive signalling of transforming growth factor (TGF)-beta. The increased TGF-beta activity in the genetic mutants was caused by accelerated turnover of the ER localized (anti-oxidant) ascorbic acid that affected collagen deposition in the extracellular matrix. In a mouse mutant of SEPN1, which is dependent on exogenous ascorbic acid, a limited intake of ascorbic acid revealed a myopathic phenotype as a consequence of an altered TGF-beta signalling. Indeed, systemic antagonism of TGF-beta re-established skeletal muscle function in SEPN1 mutant mice. In conclusion, this study sheds new light on the molecular mechanism of SEPN1-related myopathies and indicates that the TGF-beta/ERO1/ascorbic acid axis offers potential for their treatment. PMID:28106121

  5. New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum.

    PubMed

    Rudner, J; Jendrossek, V; Belka, C

    2002-10-01

    The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.

  6. Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

    NASA Technical Reports Server (NTRS)

    Hong, B.; Ichida, A.; Wang, Y.; Gens, J. S.; Pickard, B. G.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    1999-01-01

    A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

  7. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    SciTech Connect

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-07-15

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

  8. Type 2 diabetes mellitus induces congenital heart defects in murine embryos by increasing oxidative stress, endoplasmic reticulum stress, and apoptosis

    PubMed Central

    Wu, Yanqing; Reece, E. Albert; Zhong, Jianxiang; Dong, Daoyin; Shen, Wei-Bin; Harman, Christopher R.; Yang, Peixin

    2017-01-01

    BACKGROUND Maternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects remain largely unknown. OBJECTIVE We aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects. STUDY DESIGN A mouse model of maternal type 2 diabetes mellitus was established by feeding female mice a high-fat diet (60% fat). After 15 weeks on the high-fat diet, the mice showed characteristics of maternal type 2 diabetes mellitus. Control dams were either fed a normal diet (10% fat) or the high-fat diet during pregnancy only. Female mice from the high-fat diet group and the 2 control groups were mated with male mice that were fed a normal diet. At E12.5, embryonic hearts were harvested to determine the levels of lipid peroxides and superoxide, endoplasmic reticulum stress markers, cleaved caspase 3 and 8, and apoptosis. E17.5 embryonic hearts were harvested for the detection of congenital heart defect formation using India ink vessel patterning and histological examination. RESULTS Maternal type 2 diabetes mellitus significantly induced ventricular septal defects and persistent truncus arteriosus in the developing heart, along with increasing oxidative stress markers, including superoxide and lipid peroxidation; endoplasmic reticulum stress markers, including protein levels of phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase, phosphorylated-IRE1α, phosphorylated-eIF2α, C/EBP homologous protein, and binding immunoglobulin

  9. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

    2014-12-01

    Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung

  10. Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress

    PubMed Central

    Liu, Mi; Xue, Mei; Wang, Xiao-Reng; Tao, Tian-Qi; Xu, Fei-Fei; Liu, Xiu-Hua; Shi, Da-Zhuo

    2015-01-01

    Background Endoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings

  11. Fluoride-elicited developmental testicular toxicity in rats: Roles of endoplasmic reticulum stress and inflammatory response

    SciTech Connect

    Zhang, Shun; Jiang, Chunyang; Liu, Hongliang; Guan, Zhizhong; Zeng, Qiang; Zhang, Cheng; Lei, Rongrong; Xia, Tao; Gao, Hui; Yang, Lu; Chen, Yihu; Wu, Xue; Zhang, Xiaofei; Cui, Yushan; Yu, Linyu; Wang, Zhenglun; Wang, Aiguo

    2013-09-01

    Long-term excessive fluoride intake is known to be toxic and can damage a variety of organs and tissues in the human body. However, the molecular mechanisms underlying fluoride-induced male reproductive toxicity are not well understood. In this study, we used a rat model to simulate the situations of human exposure and aimed to evaluate the roles of endoplasmic reticulum (ER) stress and inflammatory response in fluoride-induced testicular injury. Sprague–Dawley rats were administered with sodium fluoride (NaF) at 25, 50 and 100 mg/L via drinking water from pre-pregnancy to gestation, birth and finally to post-puberty. And then the testes of male offspring were studied at 8 weeks of age. Our results demonstrated that fluoride treatment increased MDA accumulation, decreased SOD activity, and enhanced germ cell apoptosis. In addition, fluoride elevated mRNA and protein levels of glucose-regulated protein 78 (GRP78), inositol requiring ER-to-nucleus signal kinase 1 (IRE1), and C/EBP homologous protein (CHOP), indicating activation of ER stress signaling. Furthermore, fluoride also induced testicular inflammation, as manifested by gene up-regulation of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in a nuclear factor-κB (NF-κB)-dependent manner. These were associated with marked histopathological lesions including injury of spermatogonia, decrease of spermatocytes and absence of elongated spermatids, as well as severe ultrastructural abnormalities in testes. Taken together, our results provide compelling evidence that ER stress and inflammation would be novel and significant mechanisms responsible for fluoride-induced disturbance of spermatogenesis and germ cell loss in addition to oxidative stress. - Highlights: • We used a rat model to simulate the situations of human fluoride (F) exposure. • Developmental F exposure induces testicular damage related with oxidative stress.

  12. The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

    SciTech Connect

    Fujimoto, Michiko; Hayashi, Teruo; Su, Tsung-Ping

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer The endoplasmic reticulum subdomain termed MAM associates with mitochondria. Black-Right-Pointing-Pointer The biophysical role of lipids in the MAM-mitochondria association is unknown. Black-Right-Pointing-Pointer The in vitro membrane association assay was used to examine the role of lipids. Black-Right-Pointing-Pointer Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP{sub 3} receptor-mediated Ca{sup 2+} influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-{beta}-cyclodextrin (M{beta}C) significantly increases the association between MAMs and mitochondria, whereas M{beta}C saturated with cholesterol does not change the association. {sup 14}C-Serine pulse-labeling demonstrated that the treatment of living cells with M{beta}C decreases the level of de novo synthesized {sup 14}C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of

  13. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    PubMed Central

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

  14. Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

    PubMed Central

    Skehel, P. A.; Fabian-Fine, R.; Kandel, E. R.

    2000-01-01

    VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum–resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis. PMID:10655491

  15. The endoplasmic reticulum stress response in aging and age-related diseases

    PubMed Central

    Brown, Marishka K.; Naidoo, Nirinjini

    2012-01-01

    The endoplasmic reticulum(ER) is a multifunctional organelle within which protein folding, lipid biosynthesis, and calcium storage occurs. Perturbations such as energy or nutrient depletion, disturbances in calcium or redox status that disrupt ER homeostasis lead to the misfolding of proteins, ER stress and up-regulation of several signaling pathways coordinately called the unfolded protein response (UPR). The UPR is characterized by the induction of chaperones, degradation of misfolded proteins and attenuation of protein translation. The UPR plays a fundamental role in the maintenance of cellular homeostasis and thus is central to normal physiology. However, sustained unresolved ER stress leads to apoptosis. Aging linked declines in expression and activity of key ER molecular chaperones and folding enzymes compromise proper protein folding and the adaptive response of the UPR. One mechanism to explain age associated declines in cellular functions and age-related diseases is a progressive failure of chaperoning systems. In many of these diseases, proteins or fragments of proteins convert from their normally soluble forms to insoluble fibrils or plaques that accumulate in a variety of organs including the liver, brain or spleen. This group of diseases, which typically occur late in life includes Alzheimer's, Parkinson's, type II diabetes and a host of less well known but often equally serious conditions such as fatal familial insomnia. The UPR is implicated in many of these neurodegenerative and familial protein folding diseases as well as several cancers and a host of inflammatory diseases including diabetes, atherosclerosis, inflammatory bowel disease and arthritis. This review will discuss age-related changes in the ER stress response and the role of the UPR in age-related diseases. PMID:22934019

  16. Mitochondrial Dysregulation Secondary to Endoplasmic Reticulum Stress in Autosomal Dominant Tubulointerstitial Kidney Disease – UMOD (ADTKD-UMOD)

    PubMed Central

    Kemter, Elisabeth; Fröhlich, Thomas; Arnold, Georg J.; Wolf , Eckhard; Wanke, Rüdiger

    2017-01-01

    ‘Autosomal dominant tubulointerstitial kidney disease – UMOD’ (ADTKD-UMOD) is caused by impaired maturation and secretion of mutant uromodulin (UMOD) in thick ascending limb of Henle loop (TAL) cells, resulting in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). To gain insight into pathophysiology, we analysed proteome profiles of TAL-enriched outer renal medulla samples from ADTKD-UMOD and control mice by quantitative LC-MS/MS. In total, 212 differentially abundant proteins were identified. Numerous ER proteins, including BiP (HSPA5), phosphorylated eIF2α (EIF2S1), ATF4, ATF6 and CHOP (DDIT3), were increased abundant, consistent with UPR. The abundance of hypoxia-inducible proteins with stress survival functions, i.e. HYOU1, TXNDC5 and ERO1L, was also increased. TAL cells in ADTKD-UMOD showed a decreased proportion of mitochondria and reduced abundance of multiple mitochondrial proteins, associated with disturbed post-translational processing and activation of the mitochondrial transcription factor NRF1. Impaired fission of organelles, as suggested by reduced abundance of FIS1, may be another reason for disturbed biogenesis of mitochondria and peroxisomes. Reduced amounts of numerous proteins of the OXPHOS and citrate cycle pathways, and activation of the LKB1-AMPK-pathway, a sensor pathway of cellular energy deficits, suggest impaired energy homeostasis. In conclusion, our study revealed secondary mitochondrial dysfunction in ADTKD-UMOD. PMID:28220896

  17. Glycation of paraoxonase 1 by high glucose instigates endoplasmic reticulum stress to induce endothelial dysfunction in vivo

    PubMed Central

    Yu, Wei; Liu, Xiaoli; Feng, Liru; Yang, Hui; Yu, Weiye; Feng, Tiejian; Wang, Shuangxi; Wang, Jun; Liu, Ning

    2017-01-01

    High-density lipoprotein (HDL) modulates low-density lipoprotein and cell membrane oxidation through the action of paraoxonase-1 (PON1). Endoplasmic reticulum (ER) stress has been linked to a wide range of human pathologies including diabetes, obesity, and atherosclerosis. Previous studies have reported that PON1 is glycated in diabetes. The aim of this study is to investigate whether and how PON1 glycation contributes to endothelial dysfunction in diabetes. ER stress markers were monitored by western blot. Endothelial function was determined by organ bath. Incubation of recombinant PON1 proteins with high glucose increased PON1 glycation and reduced PON1 activity. Exposure of HUVECs to glycated PON1 induced prolonged ER stress and reduced SERCA activity, which were abolished by tempol, apocynin, BAPTA, and p67 and p22 siRNAs. Chronic administration of amino guanidine or 4-PBA prevented endothelial dysfunction in STZ-injected rats. Importantly, injection of glycated PON1 but not native PON1 induced aberrant ER stress and endothelial dysfunction in rats, which were attenuated by tempol, BAPTA, and 4-PBA. In conclusion, glycation of PON1 by hyperglycemia induces endothelial dysfunction through ER stress. In perspectives, PON1 glycation is a novel risk factor of hyperglycemia-induced endothelial dysfunction. Therefore, inhibition of oxidative stress, chelating intracellular Ca2+, and ER chaperone would be considered to reduce vascular complications in diabetes. PMID:28374834

  18. Endoplasmic Reticulum Retention Signal-Dependent Glycylation of the Hsp70/Grp170-Related Pgp1p in Tetrahymena▿ †

    PubMed Central

    Xie, Rong; Clark, Kathleen M.; Gorovsky, Martin A.

    2007-01-01

    Glycylation is an uncommon posttranslational modification. It has been found that tubulin glycylation is essential for cell survival in Tetrahymena. Here we describe PGP1, a Tetrahymena gene encoding an Hsp70 homologue that is a novel glycylated protein. Pgp1p is a conserved glycoprotein that localizes within the lumen of the endoplasmic reticulum (ER). We demonstrate that PGP1 is essential for viability and present evidence that both glycosylation and ER retention are necessary but not sufficient for glycylation. PMID:17189490

  19. Neuropeptide accretions in the endoplasmic reticulum of oxytocinergic neurons in cats, monkeys and rabbits: a widespread phenomenon.

    PubMed Central

    Pow, D V

    1992-01-01

    Light microscopic observations using Nomarski optics on the aldehyde-fixed hypothalamus of normal adult cats, monkeys and rabbits revealed the presence of cells in the supraoptic, paraventricular and periventricular nuclei which possessed yellow birefringent inclusions. Immunogold labelling showed that in each species the cells displayed oxytocin-like immunoreactivity, both in electron-dense inclusions within some (but not all) cisterns of rough endoplasmic reticulum and in secretory granules. The cells in cats and rabbits were in all respects indistinguishable from the homologous 'birefringent' cells previously described in rats, but in monkeys, cells frequently contained additional inclusions in cisterns of rough endoplasmic reticulum which did not display oxytocin or vasopressin-like immunoreactivity, even after trypsin, pepsin or chymotrypsin treatment of sections. Observations on cats and rabbits using fluorescence microscopy revealed that the birefringent cells possessed bright autofluorescence which facilitated the identification of more cells than were seen using Nomarski optics alone. Autofluorescence was abolished when sections were mounted in glycerol, or when exposed to light for protracted periods of time. Attempts to label for monoamines in these cells were not successful, suggesting that the fluorescence is not due to aldehyde-induced amine fluorescence. It is not clear why neuropeptides are retained in some rough endoplasmic reticulum cisterns. It is possible that these birefringent cells contain a peptide, or peptides, which are abnormal in some manner, or which may be other members of the oxytocin gene family. Alternatively, the processing of neuropeptides to permit their export to the Golgi apparatus may be deficient. Acetylcholinesterase (AChE) histochemistry revealed that, unlike other oxytocin neurons, cells with intracellular accretions lacked detectable acetyl cholinesterase. As AChE is a known peptidase, it may be involved in regulating

  20. C1q/TNF-Related Protein 9 Protects Diabetic Rat Heart against Ischemia Reperfusion Injury: Role of Endoplasmic Reticulum Stress

    PubMed Central

    Bai, Sanxing; Cheng, Liang; Yang, Yang; Fan, Chongxi; Zhao, Dajun; Qin, Zhigang; Feng, Xiao; Zhao, Lin; Ma, Jipeng; Wang, Xiaowu; Yang, Jian; Xu, Xuezeng

    2016-01-01

    As a newly identified adiponectin paralog, C1q/TNF-related protein 9 (CTRP9) reduces myocardial ischemia reperfusion (IR) injury through partially understood mechanisms. In the present study, we sought to identify the role of endoplasmic reticulum stress (ERS) in CTRP9 induced cardioprotection in diabetic heart. Isolated hearts from high-fat-diet (HFD) induced type 2 diabetic Sprague-Dawley rats were subjected to ex vivo IR protocol via a Langendorff apparatus at the presence of globular CTRP9. CTRP9 significantly improved post-IR heart function and reduced cardiac infarction, cardiomyocytes apoptosis, Caspase-3, Caspase-9, Caspase-12, TNF-α expression, and lactate dehydrogenase activity. The cardioprotective effect of CTRP9 was associated with reduced ERS and increased expression of disulfide-bond A oxidoreductase-like protein (DsbA-L) in diabetic heart. CTRP9 reduced ERS in thapsigargin (TG) treated cardiomyocytes and protected endoplasmic reticulum (ER) stressed H9c2 cells against simulated ischemia reperfusion (SIR) injury, concurrent with increased expression of DsbA-L. Knockdown of DsbA-L increased ERS and attenuated CTRP9 induced protection against SIR injury in H9c2 cells. Our findings demonstrated for the first time that CTRP9 exerts cardioprotection by reducing ERS in diabetic heart through increasing DsbA-L. PMID:27795806

  1. Vesicular Trafficking of Incoming Human Papillomavirus 16 to the Golgi Apparatus and Endoplasmic Reticulum Requires γ-Secretase Activity

    PubMed Central

    Zhang, Wei; Kazakov, Teymur; Popa, Andreea

    2014-01-01

    ABSTRACT The route taken by papillomaviruses from the cell surface to the nucleus during infection is incompletely understood. Here, we developed a novel human papillomavirus 16 (HPV16) pseudovirus in which the carboxy terminus of the minor capsid protein L2 is exposed on the exterior of the intact capsid prior to cell binding. With this pseudovirus, we used the proximity ligation assay immune detection technique to demonstrate that during entry HPV16 L2 traffics into and out of the early endosome prior to Golgi localization, and we demonstrated that L2 enters the endoplasmic reticulum during entry. The cellular membrane-associated protease, γ-secretase, is required for infection by HPV16 pseudovirus and authentic HPV16. We also showed that inhibition of γ-secretase does not interfere substantively with virus internalization, initiation of capsid disassembly, entry into the early endosome, or exit from this compartment, but γ-secretase is required for localization of L2 and viral DNA to the Golgi apparatus and the endoplasmic reticulum. These results show that incoming HPV16 traffics sequentially from the cell surface to the endosome and then to the Golgi apparatus and the endoplasmic reticulum prior to nuclear entry. PMID:25227470

  2. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    PubMed

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-05

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  3. Dysfunction in endoplasmic reticulum-mitochondria crosstalk underlies SIGMAR1 loss of function mediated motor neuron degeneration.

    PubMed

    Bernard-Marissal, Nathalie; Médard, Jean-Jacques; Azzedine, Hamid; Chrast, Roman

    2015-04-01

    Mutations in Sigma 1 receptor (SIGMAR1) have been previously identified in patients with amyotrophic lateral sclerosis and disruption of Sigmar1 in mouse leads to locomotor deficits. However, cellular mechanisms underlying motor phenotypes in human and mouse with disturbed SIGMAR1 function have not been described so far. Here we used a combination of in vivo and in vitro approaches to investigate the role of SIGMAR1 in motor neuron biology. Characterization of Sigmar1(-/-) mice revealed that affected animals display locomotor deficits associated with muscle weakness, axonal degeneration and motor neuron loss. Using primary motor neuron cultures, we observed that pharmacological or genetic inactivation of SIGMAR1 led to motor neuron axonal degeneration followed by cell death. Disruption of SIGMAR1 function in motor neurons disturbed endoplasmic reticulum-mitochondria contacts, affected intracellular calcium signalling and was accompanied by activation of endoplasmic reticulum stress and defects in mitochondrial dynamics and transport. These defects were not observed in cultured sensory neurons, highlighting the exacerbated sensitivity of motor neurons to SIGMAR1 function. Interestingly, the inhibition of mitochondrial fission was sufficient to induce mitochondria axonal transport defects as well as axonal degeneration similar to the changes observed after SIGMAR1 inactivation or loss. Intracellular calcium scavenging and endoplasmic reticulum stress inhibition were able to restore mitochondrial function and consequently prevent motor neuron degeneration. These results uncover the cellular mechanisms underlying motor neuron degeneration mediated by loss of SIGMAR1 function and provide therapeutically relevant insight into motor neuronal diseases.

  4. Re-evaluation of the Role of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) in Cellular Calcium Signaling*

    PubMed Central

    Lin-Moshier, Yaping; Sebastian, Peter J.; Higgins, LeeAnn; Sampson, Natalie D.; Hewitt, Jane E.; Marchant, Jonathan S.

    2013-01-01

    Changes in cytoplasmic Ca2+ concentration, resulting from activation of intracellular Ca2+ channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca2+ homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca2+ channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca2+ channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP3Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest. PMID:23148228

  5. Myopathy in Marinesco-Sjögren syndrome links endoplasmic reticulum chaperone dysfunction to nuclear envelope pathology.

    PubMed

    Roos, Andreas; Buchkremer, Stephan; Kollipara, Laxmikanth; Labisch, Thomas; Gatz, Christian; Zitzelsberger, Manuela; Brauers, Eva; Nolte, Kay; Schröder, J Michael; Kirschner, Janbernd; Jesse, Christopher Marvin; Goebel, Hans Hilmar; Goswami, Anand; Zimmermann, Richard; Zahedi, René Peiman; Senderek, Jan; Weis, Joachim

    2014-05-01

    Marinesco-Sjögren syndrome (MSS) features cerebellar ataxia, mental retardation, cataracts, and progressive vacuolar myopathy with peculiar myonuclear alterations. Most MSS patients carry homozygous or compound heterozygous SIL1 mutations. SIL1 is a nucleotide exchange factor for the endoplasmic reticulum resident chaperone BiP which controls a plethora of essential processes in the endoplasmic reticulum. In this study we made use of the spontaneous Sil1 mouse mutant woozy to explore pathomechanisms leading to Sil1 deficiency-related skeletal muscle pathology. We found severe, progressive myopathy characterized by alterations of the sarcoplasmic reticulum, accumulation of autophagic vacuoles, mitochondrial changes, and prominent myonuclear pathology including nuclear envelope and nuclear lamina alterations. These abnormalities were remarkably similar to the myopathy in human patients with MSS. In particular, the presence of perinuclear membranous structures which have been reported as an ultrastructural hallmark of MSS-related myopathy could be confirmed in woozy muscles. We found that these structures are derived from the nuclear envelope and nuclear lamina and associate with proliferations of the sarcoplasmic reticulum. In line with impaired function of BiP secondary to loss of its nucleotide exchange factor Sil1, we observed activation of the unfolded protein response and the endoplasmic-reticulum-associated protein degradation-pathway. Despite initiation of the autophagy-lysosomal system, autophagic clearance was found ineffective which is in agreement with the formation of autophagic vacuoles. This report identifies woozy muscle as a faithful phenocopy of the MSS myopathy. Moreover, we provide a link between two well-established disease mechanisms in skeletal muscle, dysfunction of chaperones and nuclear envelope pathology.

  6. Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

    PubMed Central

    Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

    2012-01-01

    The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

  7. Changes in ribbon synapses and rough endoplasmic reticulum of rat utricular macular hair cells in weightlessness

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    2000-01-01

    This study combined ultrastructural and statistical methods to learn the effects of weightlessness on rat utricular maculae. A principle aim was to determine whether weightlessness chiefly affects ribbon synapses of type II cells, since the cells communicate predominantly with branches of primary vestibular afferent endings. Maculae were microdissected from flight and ground control rat inner ears collected on day 13 of a 14-day spaceflight (F13), landing day (R0) and day 14 postflight (R14) and were prepared for ultrastructural study. Ribbon synapses were counted in hair cells examined in a Zeiss 902 transmission electron microscope. Significance of synaptic mean differences was determined for all hair cells contained within 100 section series, and for a subset of complete hair cells, using SuperANOVA software. The synaptic mean for all type II hair cells of F13 flight rats increased by 100%, and that for complete cells by 200%. Type I cells were less affected, with synaptic mean differences statistically insignificant in complete cells. Synapse deletion began within 8 h upon return to Earth. Additionally, hair cell laminated rough endoplasmic reticulum of flight rats was reversibly disorganized on R0. Results support the thesis that synapses in type II hair cells are uniquely affected by altered gravity. Type II hair cells may be chiefly sensors of gravitational and type I cells of translational linear accelerations.

  8. ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes.

    PubMed

    Liu, Na; Stoica, George; Yan, Mingshan; Scofield, Virginia L; Qiang, Wenan; Lynn, William S; Wong, Paul K Y

    2005-12-01

    ATM kinase, the product of the ataxia telangiectasia mutated (Atm) gene, is activated by genomic damage. ATM plays a crucial role in cell growth and development. Here we report that primary astrocytes isolated from ATM-deficient mice grow slowly, become senescent, and die in culture. However, before reaching senescence, these primary Atm(-/-) astrocytes, like Atm(-/-) lymphocytes, show increased spontaneous DNA synthesis. These astrocytes also show markers of oxidative stress and endoplasmic reticulum (ER) stress, including increased levels of heat shock proteins (HSP70 and GRP78), malondialdehyde adducts, Cu/Zn superoxide dismutase, procaspase 12 cleavage, and redox-sensitive phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In addition, HSP70 and ERK1/2 phosphorylation are upregulated in the cerebella of ATM-deficient mice. This increase in ERK1/2 phosphorylation is seen primarily in cerebellar astrocytes, or Bergmann glia, near degenerating Purkinje cells. ERK1/2 activation and astrogliosis are also found in other parts of the brain, for example, the cortex. We conclude that ATM deficiency induces intrinsic growth defects, oxidative stress, ER stress, and ERKs activation in astrocytes.

  9. Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways

    PubMed Central

    Werstuck, Geoff H.; Lentz, Steven R.; Dayal, Sanjana; Hossain, Gazi S.; Sood, Sudesh K.; Shi, Yuan Y.; Zhou, Ji; Maeda, Nobuyo; Krisans, Skaidrite K.; Malinow, M. Rene; Austin, Richard C.

    2001-01-01

    Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine β-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element–binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia. PMID:11375416

  10. A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum

    SciTech Connect

    Ganan, S.; Cazzulo, J.J.; Parodi, A.J. )

    1991-03-26

    N-Linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc{sub 1}Man{sub 5-9}GlcNAc{sub 2} and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man{sub 9}GlcNAc{sub 2} in protein N-glycosylation) were grown in the presence of ({sup 14}C)glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1,000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. No evidence for the presence of an endomannosidase yielding GlcMan from the glucosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-33% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.

  11. Green fluorescent protein-based monitoring of endoplasmic reticulum redox poise

    PubMed Central

    Birk, Julia; Ramming, Thomas; Odermatt, Alex; Appenzeller-Herzog, Christian

    2013-01-01

    Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive oxidants, which can be both upstream and downstream of ER stress. Accordingly, detrimental intracellular stress signals are amplified through establishment of a vicious cycle. An increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. Experimental monitoring of stress-induced, time-resolved changes in ER reduction-oxidation (redox) states is therefore important. Organelle-specific examination of redox changes has been facilitated by the advent of genetically encoded, fluorescent probes, which can be targeted to different subcellular locations by means of specific amino acid extensions. These probes include redox-sensitive green fluorescent proteins (roGFPs) and the yellow fluorescent protein-based redox biosensor HyPer. In the case of roGFPs, variants with known specificity toward defined redox couples are now available. Here, we review the experimental framework to measure ER redox changes using ER-targeted fluorescent biosensors. Advantages and drawbacks of plate-reader and microscopy-based measurements are discussed, and the power of these techniques demonstrated in the context of selected cell culture models for ER stress. PMID:23781233

  12. Endoplasmic Reticulum Thiol Oxidase Deficiency Leads to Ascorbic Acid Depletion and Noncanonical Scurvy in Mice

    PubMed Central

    Zito, Ester; Hansen, Henning Gram; Yeo, Giles S.H.; Fujii, Junichi; Ron, David

    2012-01-01

    Summary Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy. PMID:22981861

  13. Role of Mitochondria-Associated Endoplasmic Reticulum Membrane in Inflammation-Mediated Metabolic Diseases.

    PubMed

    Thoudam, Themis; Jeon, Jae-Han; Ha, Chae-Myeong; Lee, In-Kyu

    2016-01-01

    Inflammation is considered to be one of the most critical factors involved in the development of complex metabolic diseases such as type 2 diabetes, cancer, and cardiovascular disease. A few decades ago, the discovery of mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) was followed by the identification of its roles in regulating cellular homeostatic processes, ranging from cellular bioenergetics to apoptosis. MAM provides an excellent platform for numerous signaling pathways; among them, inflammatory signaling pathways associated with MAM play a critical role in cellular defense during pathogenic infections and metabolic disorders. However, induction of MAM causes deleterious effects by amplifying mitochondrial reactive oxygen species generation through increased calcium transfer from the ER to mitochondria, thereby causing mitochondrial damage and release of mitochondrial components into the cytosol as damage-associated molecular patterns (DAMPs). These mitochondrial DAMPs rapidly activate MAM-resident inflammasome components and other inflammatory factors, which promote inflammasome complex formation and release of proinflammatory cytokines in pathological conditions. Long-term stimulation of the inflammasome instigates chronic inflammation, leading to the pathogenesis of metabolic diseases. In this review, we summarize the current understanding of MAM and its association with inflammation-mediated metabolic diseases.

  14. Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

    PubMed Central

    Jiang, Chunping; Luo, Dongjun; Cao, Yin; Liu, Baorui

    2014-01-01

    Background. Hepatocellular carcinoma (HCC) remains a disastrous disease and the treatment for HCC is rather limited. Separation and identification of active compounds from traditionally used herbs in HCC treatment may shed light on novel therapeutic drugs for HCC. Methods. Cell viability and colony forming assay were conducted to determine anti-HCC activity. Morphology of cells and activity of caspases were analyzed. Antiapoptotic Bcl-2 family proteins and JNK were also examined. Levels of unfolded protein response (UPR) markers were determined and intracellular calcium was assayed. Small interfering RNAs (siRNAs) were used to investigate the role of UPR and autophagy in baicalein-induced cell death. Results. Among four studied flavonoids, only baicalein exhibited satisfactory inhibition of viability and colony formation of HCC cells within water-soluble concentration. Baicalein induced apoptosis via endoplasmic reticulum (ER) stress, possibly by downregulating prosurvival Bcl-2 family, increasing intracellular calcium, and activating JNK. CHOP was the executor of cell death during baicalein-induced ER stress while eIF2α and IRE1α played protective roles. Protective autophagy was also triggered by baicalein in HCC cells. Conclusion. Baicalein exhibits prominent anti-HCC activity. This flavonoid induces apoptosis and protective autophagy via ER stress. Combination of baicalein and autophagy inhibitors may represent a promising therapy against HCC. PMID:24995326

  15. Protective effect of mild endoplasmic reticulum stress on radiation-induced bystander effects in hepatocyte cells.

    PubMed

    Xie, Yuexia; Ye, Shuang; Zhang, Jianghong; He, Mingyuan; Dong, Chen; Tu, Wenzhi; Liu, Peifeng; Shao, Chunlin

    2016-12-13

    Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the defense and self-protective mechanisms of bystander normal cells are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 cells under either normoxia or hypoxia, where the ratio of the yield of bystander MN induction to the yield of radiation-induced MN formation under hypoxia was much higher than that of normoxia. Nonetheless, thapsigargin induced endoplasmic reticulum (ER) stress and dramatically suppressed this bystander response manifested as the decrease of MN and apoptosis inductions. Meanwhile, the interference of BiP gene, a major ER chaperone, amplified the detrimental RIBE. More precisely, thapsigargin provoked ER sensor of PERK to initiate an instantaneous and moderate ER stress thus defensed the hazard form RIBE, while BiP depletion lead to persistently destroyed homeostasis of ER and exacerbated cell injury. These findings provide new insights that the mild ER stress through BiP-PERK-p-eIF2α signaling pathway has a profound role in protecting cellular damage from RIBE and hence may decrease the potential secondary cancer risk after cancer radiotherapy.

  16. Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5

    PubMed Central

    Vogiatzi, Fotini; Brandt, Dominique T.; Schneikert, Jean; Fuchs, Jeannette; Grikscheit, Katharina; Wanzel, Michael; Pavlakis, Evangelos; Charles, Joël P.; Timofeev, Oleg; Nist, Andrea; Mernberger, Marco; Kantelhardt, Eva J.; Siebolts, Udo; Bartel, Frank; Jacob, Ralf; Rath, Ariane; Moll, Roland; Grosse, Robert; Stiewe, Thorsten

    2016-01-01

    Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5′-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment. PMID:27956623

  17. Evidence for Increased Response to Induced Endoplasmic Reticulum Stress in Myeloid Cells in Acquired Aplastic Anemia.

    PubMed

    Sidhu, Alpa; Callaghan, Michael U; Gadgeel, Manisha S; Buck, Steven A; Fribley, Andrew M; Savaşan, Süreyya

    2017-04-01

    Autoimmune response targeting the hematopoietic stem cells highlights the current understanding of acquired aplastic anemia (AAA) pathogenesis. Upregulation of the unfolded protein response is the cell's rejoinder to a variety of stresses, which either result in restoring homeostasis or cell death by increased expression of the transcription factor C/EBP homologous protein. We hypothesized that there is an inherent increased sensitivity to various cellular stressors, including the ones that target endoplasmic reticulum (ER) in AAA leading to a decreased proliferation and potentially contributing to susceptibility to autologous cytotoxicity. Using archived bone marrow aspirate samples, we demonstrate that the culture-expanded AAA myeloid cells have an increased response to ER stress induced by tunicamycin leading to decreased cell proliferation. Within the AAA myeloid samples, we show that the disease status, active versus response to therapy at the time of sampling does not alter the ER stress response. This is the first report, which provides evidence for an inherent defective stress control in the myeloid cells as a possible mechanism of evolution of the disease process in AAA.

  18. Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection

    PubMed Central

    Feger, Bryan J.; Thompson, J. Will; Dubois, Laura G.; Kommaddi, Reddy P.; Foster, Matthew W.; Mishra, Rajashree; Shenoy, Sudha K.; Shibata, Yoichiro; Kidane, Yared H.; Moseley, M. Arthur; Carnell, Lisa S.; Bowles, Dawn E.

    2016-01-01

    On Earth, biological systems have evolved in response to environmental stressors, interactions dictated by physical forces that include gravity. The absence of gravity is an extreme stressor and the impact of its absence on biological systems is ill-defined. Astronauts who have spent extended time under conditions of minimal gravity (microgravity) experience an array of biological alterations, including perturbations in cardiovascular function. We hypothesized that physiological perturbations in cardiac function in microgravity may be a consequence of alterations in molecular and organellar dynamics within the cellular milieu of cardiomyocytes. We used a combination of mass spectrometry-based approaches to compare the relative abundance and turnover rates of 848 and 196 proteins, respectively, in rat neonatal cardiomyocytes exposed to simulated microgravity or normal gravity. Gene functional enrichment analysis of these data suggested that the protein content and function of the mitochondria, ribosomes, and endoplasmic reticulum were differentially modulated in microgravity. We confirmed experimentally that in microgravity protein synthesis was decreased while apoptosis, cell viability, and protein degradation were largely unaffected. These data support our conclusion that in microgravity cardiomyocytes attempt to maintain mitochondrial homeostasis at the expense of protein synthesis. The overall response to this stress may culminate in cardiac muscle atrophy. PMID:27670941

  19. Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

    PubMed Central

    Davis, Lianne C.; Wagner, Siegfried K.T.Y.; Lewis, Alexander M.; Parrington, John; Churchill, Grant C.

    2013-01-01

    The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca2+ stores that release Ca2+ in response to the second messengers IP3 and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca2+ released from acidic organelles by NAADP subsequently recruits IP3 or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca2+ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca2+ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca2+-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca2+ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca2+ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca2+ oscillations and waves. PMID:23479744

  20. Endoplasmic Reticulum PI(3)P lipid binding targets malaria proteins to the host cell

    PubMed Central

    Bhattacharjee, Souvik; Stahelin, Robert V.; Speicher, Kaye D.; Speicher, David W.; Haldar, Kasturi

    2011-01-01

    SUMMARY Hundreds of effector proteins of the human malaria parasite Plasmodium falciparum constitute a `secretome', carrying a host-targeting (HT) signal, which predicts their export from the intracellular pathogen into the surrounding erythrocyte. Cleavage of the HT signal by a parasite endoplasmic reticulum (ER) protease, plasmepsin V, is the proposed export mechanism. Here we show that the HT signal exports by recognition of the lipid phosphatidylinositol-3-phosphate (PI(3)P) in the ER, prior to and independent of protease action. Secretome HT signals, including those of major virulence determinants bind PI(3)P with nanomolar affinity and amino acid specificities displayed by HT-mediated export. PI(3)P-enriched regions are detected within the parasite's ER, co-localize with endogenous HT signal on ER precursors, which also display high affinity binding to PI(3)P. A related, pathogenic oomycete's HT signal export is dependent on PI(3)P binding, without cleavage by plasmepsin V. Thus PI(3)P in the ER functions in mechanisms of secretion and pathogenesis. PMID:22265412

  1. Characterization of an Endoplasmic Reticulum-associated Silaffin Kinase from the Diatom Thalassiosira pseudonana*

    PubMed Central

    Sheppard, Vonda; Poulsen, Nicole; Kröger, Nils

    2010-01-01

    The formation of SiO2-based cell walls by diatoms (a large group of unicellular microalgae) is a well established model system for the study of molecular mechanisms of biological mineral morphogenesis (biomineralization). Diatom biomineralization involves highly phosphorylated proteins (silaffins and silacidins), analogous to other biomineralization systems, which also depend on diverse sets of phosphoproteins (e.g. mammalian teeth and bone, mollusk shells, and sponge silica). The phosphate moieties on biomineralization proteins play an essential role in mineral formation, yet the kinases catalyzing the phosphorylation of these proteins have remained poorly characterized. Recent functional genomics studies on the diatom Thalassiosira pseudonana have revealed >100 proteins potentially involved in diatom silica formation. Here we have characterized the biochemical properties and biological function of one of these proteins, tpSTK1. Multiple tpSTK1-like proteins are encoded in diatom genomes, all of which exhibit low but significant sequence similarity to kinases from other organisms. We show that tpSTK1 has serine/threonine kinase activity capable of phosphorylating silaffins but not silacidins. Cell biological and biochemical analysis demonstrated that tpSTK1 is an abundant component of the lumen of the endoplasmic reticulum. The present study provides the first molecular structure of a kinase that appears to catalyze phosphorylation of biomineral forming proteins in vivo. PMID:19889629

  2. Endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate

    PubMed Central

    Brookes, Steven J.; Barron, Martin J.; Boot-Handford, Ray; Kirkham, Jennifer; Dixon, Michael J.

    2014-01-01

    Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease. PMID:24362885

  3. Peroxisomes, lipid droplets, and endoplasmic reticulum “hitchhike” on motile early endosomes

    PubMed Central

    Guimaraes, Sofia C.; Schuster, Martin; Bielska, Ewa; Dagdas, Gulay; Kilaru, Sreedhar; Meadows, Ben R.A.; Schrader, Michael

    2015-01-01

    Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. PMID:26620910

  4. Endoplasmic reticulum targeting in Ewing's sarcoma by the alkylphospholipid analog edelfosine

    PubMed Central

    Bonilla, Ximena; Dakir, EL-Habib; Mollinedo, Faustino; Gajate, Consuelo

    2015-01-01

    Ewing's sarcoma (ES) is the second most common bone cancer in children and young people. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the prototype of a family of synthetic antitumor compounds, collectively known as alkylphospholipid analogs (APLs). We have found that APLs ranked edelfosine>perifosine>erucylphosphocholine>miltefosine for their capacity to promote apoptosis in ES cells. Edelfosine accumulated in the endoplasmic reticulum (ER) and triggered an ER stress response that eventually led to caspase-dependent apoptosis in ES cells. This apoptotic response involved mitochondrial-mediated processes, with cytochrome c release, caspase-9 activation and generation of reactive oxygen species. Edelfosine-induced apoptosis was also dependent on sustained c-Jun NH2-terminal kinase activation. Oral administration of edelfosine showed a potent in vivo antitumor activity in an ES xenograft animal model. Histochemical staining gave evidence for ER stress response and apoptosis in the ES tumors isolated from edelfosine-treated mice. Edelfosine showed a preferential action on ES tumor cells as compared to non-transformed osteoblasts, and appeared to be well suited for combination therapy regimens. These results demonstrate in vitro and in vivo antitumor activity of edelfosine against ES cells that is mediated by caspase activation and ER stress, and provide the proof of concept for a putative edelfosine- and ER stress-mediated approach forES treatment. PMID:25999349

  5. Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

    PubMed Central

    Bertrand, Luc

    2015-01-01

    Increasing evidence demonstrates that the antiretroviral drugs (ARVds) used for human immunodeficiency virus (HIV) treatment have toxic effects that result in various cellular and tissue pathologies; however, their impact on the cells composing the blood-brain barrier is poorly understood. The current study focused on ARVds, used either in combination or alone, on the induction of endoplasmic reticulum (ER) stress responses in human brain endothelial cells. Among studied drugs (efavirenz, tenofovir, emtricitabine, lamivudine, and indinavir), only efavirenz increased ER stress via upregulation and activation of protein kinase-like ER kinase PERK and inositol requiring kinase 1α (IRE1α). At the same time, efavirenz diminished autophagic activity, a surprising result because typically the induction of ER stress is linked to enhanced autophagy. These results were confirmed in microvessels of HIV transgenic mice chronically administered with efavirenz. In a series of further experiments, we identified that efavirenz dysregulated ER stress and autophagy by blocking the activity of the Beclin-1/Atg14/PI3KIII complex in regard to synthesis of phosphatidylinositol 3-phosphate, a process that is linked to the formation of autophagosomes. Because autophagy is a protective mechanism involved in the removal of dysfunctional proteins and organelles, its inhibition can contribute to the toxicity of efavirenz or the development of neurodegenerative disease in HIV patients treated with this drug. PMID:25987489

  6. Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

    PubMed Central

    Kaneko, Masayuki; Iwase, Ikuko; Yamasaki, Yuki; Takai, Tomoko; Wu, Yan; Kanemoto, Soshi; Matsuhisa, Koji; Asada, Rie; Okuma, Yasunobu; Watanabe, Takeshi; Imaizumi, Kazunori; Nomura, Yausyuki

    2016-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s. PMID:27485036

  7. Quantitative organellar proteomics analysis of rough endoplasmic reticulum from normal and acute pancreatitis rat pancreas

    PubMed Central

    Chen, Xuequn; Sans, Maria Dolors; Strahler, John R.; Karnovsky, Alla; Ernst, Stephen A.; Michailidis, George; Andrews, Philip C.; Williams, John A.

    2010-01-01

    Summary The rough endoplasmic reticulum (RER) is a central organelle for synthesizing and processing digestive enzymes and alteration of ER functions may participate in the pathogenesis of acute pancreatitis (AP). To comprehensively characterize the normal and diseased RER subproteome, this study quantitatively compared the protein compositions of pancreatic RER between normal and AP animals using isobaric tags (iTRAQ) and 2D LC-MALDI-MS/MS. A total of 469 unique proteins were revealed from four independent experiments using two different AP models. These proteins belong to a large number of functional categories including ribosomal proteins, translocon subunits, chaperones, secretory proteins, and glyco- and lipid-processing enzymes. 37 RER proteins (25 unique in arginine-induced, 6 unique in caerulein-induced and 6 common in both models of AP) showed significant changes during AP including translational regulators and digestive enzymes whereas only mild changes were found in some ER chaperones. The six proteins common to both AP models including a decrease in pancreatic triacylglycerol lipase precursor, Erp27, and prolyl 4-hydroxylase beta polypeptide as well as a dramatic increase in fibrinogen alpha, beta and gamma chains. These results suggest that the early stages of AP involve changes of multiple RER proteins that may affect the synthesis and processing of digestive enzymes. PMID:19954227

  8. Indium and indium tin oxide induce endoplasmic reticulum stress and oxidative stress in zebrafish (Danio rerio).

    PubMed

    Brun, Nadja Rebecca; Christen, Verena; Furrer, Gerhard; Fent, Karl

    2014-10-07

    Indium and indium tin oxide (ITO) are extensively used in electronic technologies. They may be introduced into the environment during production, use, and leaching from electronic devices at the end of their life. At present, surprisingly little is known about potential ecotoxicological implications of indium contamination. Here, molecular effects of indium nitrate (In(NO3)3) and ITO nanoparticles were investigated in vitro in zebrafish liver cells (ZFL) cells and in zebrafish embryos and novel insights into their molecular effects are provided. In(NO3)3 led to induction of endoplasmic reticulum (ER) stress response, induction of reactive oxygen species (ROS) and induction of transcripts of pro-apoptotic genes and TNF-α in vitro at a concentration of 247 μg/L. In(NO3)3 induced the ER stress key gene BiP at mRNA and protein level, as well as atf6, which ultimately led to induction of the important pro-apoptotic marker gene chop. The activity of In(NO3)3 on ER stress induction was much stronger than that of ITO, which is explained by differences in soluble free indium ion concentrations. The effect was also stronger in ZFL cells than in zebrafish embryos. Our study provides first evidence of ER stress and oxidative stress induction by In(NO3)3 and ITO indicating a critical toxicological profile that needs further investigation.

  9. A novel transmembrane protein defines the endoplasmic reticulum stress-induced cell death pathway.

    PubMed

    Tamaki, Tomoya; Kamatsuka, Kenta; Sato, Taku; Morooka, Shuntaro; Otsuka, Kosuke; Hattori, Masahiro; Sugiyama, Tomoyasu

    2017-03-08

    Mitochondrial membrane potential (ΔΨm) maintenance is physiologically critical in cells; its loss causes apoptotic signalling and cell death. Accumulating DNA mutations and unfolded proteins in stressed cells activate signalling pathways for cell death induction. Cancer cells often fail to die even in the presence of some death signalling proteins. Here, we report a short hairpin RNA (shRNA) with an artificial sequence, denoted Psi1 shRNA, which leads to ΔΨm loss in HCT116 cells. The Psi1 shRNA target gene was shown to encode transmembrane protein 117 (TMEM117). TMEM117 knockdown led to ΔΨm loss, increased reactive oxygen species levels, up-regulation of an endoplasmic reticulum (ER) stress sensor C/EBP homologous protein and active caspase-3 expression, and cell growth impairment, altering homeostasis towards cell death. TMEM117 levels were down-regulated in response to the ER stressor thapsigargin and decreased when cells showed ΔΨm loss. These results suggested that TMEM117 RNAi allowed apoptotic cell death. Therefore, TMEM117 probably mediates the signalling of ΔΨm loss in ER stress-mediated mitochondria-mediated cell death.

  10. Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

    PubMed Central

    Labunskyy, Vyacheslav M.; Gerashchenko, Maxim V.; Delaney, Joe R.; Kaya, Alaattin; Kennedy, Brian K.; Kaeberlein, Matt; Gladyshev, Vadim N.

    2014-01-01

    Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity. PMID:24391512

  11. Z α-1 antitrypsin deficiency and the endoplasmic reticulum stress response

    PubMed Central

    Greene, Catherine M; McElvaney, Noel G

    2010-01-01

    The serine proteinase inhibitor α-1 antitrypsin (AAT) is produced principally by the liver at the rate of 2 g/d. It is secreted into the circulation and provides an antiprotease protective screen throughout the body but most importantly in the lung, where it can neutralise the activity of the serine protease neutrophil elastase. Mutations leading to deficiency in AAT are associated with liver and lung disease. The most notable is the Z AAT mutation, which encodes a misfolded variant of the AAT protein in which the glutamic acid at position 342 is replaced by a lysine. More than 95% of all individuals with AAT deficiency carry at least one Z allele. ZAAT protein is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum (ER) of hepatocytes and other AAT-producing cells. This results in a loss of function associated with decreased circulating and intrapulmonary levels of AAT. However, the misfolded protein acquires a toxic gain of function that impacts on the ER. A major function of the ER is to ensure correct protein folding. ZAAT interferes with this function and promotes ER stress responses and inflammation. Here the signalling pathways activated during ER stress in response to accumulation of ZAAT are described and therapeutic strategies that can potentially relieve ER stress are discussed. PMID:21577302

  12. Miltirone exhibits antileukemic activity by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction pathways

    PubMed Central

    Zhou, Ling; Jiang, Lifeng; Xu, Maolei; Liu, Qun; Gao, Ning; Li, Ping; Liu, E-Hu

    2016-01-01

    In this study, we investigated the effects of miltirone in human leukemia cell lines, primary leukemia cells, and nude mice U937 xenograft. Treatment of cells with miltirone resulted in apoptosis, mitochondria membrane potential (MMP) collapses, increase of Bax/Bcl-2 ratio, and cytochrome c release. Miltirone triggered the endoplasmic reticulum (ER) stress identified through several key molecules of the unfolded protein response, including phosphorylated PERK, eIF2a, GRP78, GRP94, and caspase-12. Miltrone treatment also resulted in the release of Ca2+ from the ER stores and mitochondrial Ca2+ loading in the cells. Further research revealed that miltirone resulted in dose-dependent decrease in complex III activity and elevated reactive oxygen species (ROS) production in these cells. Miltirone-induced apoptosis, dissipation of MMP and ER stress were dramatically blocked by pretreatment with antioxidant N-acetylcysteine (NAC). In contrast, treatment with ER stress inhibitor TUDCA significantly attenuated miltirone-induced ROS and apoptosis in leukemia cells. Moreover, our in vivo findings showed that administration of miltirone markedly inhibited tumor growth and induced apoptosis in U937 xenograft model with low systemic toxicity. Taken together, these findings indicate that miltirone may exert its antileukemic activity by inducing apoptosis through a ROS-dependent destructive cycle involving ER stress and mitochondrial dysfunction. PMID:26848099

  13. Ultraviolet a induces endoplasmic reticulum stress response in human dermal fibroblasts.

    PubMed

    Komori, Ryota; Taniguchi, Mai; Ichikawa, Yoshiaki; Uemura, Aya; Oku, Masaya; Wakabayashi, Sadao; Higuchi, Kazuhiko; Yoshida, Hiderou

    2012-01-01

    The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism against the accumulation of unfolded proteins in the ER (ER stress) that consists of three response pathways (the ATF6, IRE1 and PERK pathways) in mammals. These pathways regulate the transcription of ER-related genes through specific cis-acting elements, ERSE, UPRE and AARE, respectively. Because the mammalian ER stress response is markedly activated in professional secretory cells, its main function was thought to be to upregulate the capacity of protein folding in the ER in accordance with the increased synthesis of secretory proteins. Here, we found that ultraviolet A (UVA) irradiation induced the conversion of an ER-localized sensor pATF6α(P) to an active transcription factor pATF6α(N) in normal human dermal fibroblasts (NHDFs). UVA also induced IRE1-mediated splicing of XBP1 mRNA as well as PERK-mediated phosphorylation of an α subunit of eukaryotic initiation factor 2. Consistent with these observations, we found that UVA increased transcription from ERSE, UPRE and AARE elements. From these results, we concluded that UVA irradiation activates all branches of the mammalian ER stress response in NHDFs. This suggests that the mammalian ER stress response is activated by not only intrinsic stress but also environmental stress.

  14. Inhalation of titanium dioxide induces endoplasmic reticulum stress-mediated autophagy and inflammation in mice.

    PubMed

    Yu, Kyeong-Nam; Sung, Jae Hyuck; Lee, Somin; Kim, Ji-Eun; Kim, Sanghwa; Cho, Won-Young; Lee, Ah Young; Park, Soo Jin; Lim, Joohyun; Park, Changhoon; Chae, Chanhee; Lee, Jin Kyu; Lee, Jinkyu; Kim, Jun-Sung; Cho, Myung-Haing

    2015-11-01

    Titanium dioxide (TiO2) nanoparticles are widely used in cosmetics, sunscreen, electronics, drug delivery systems, and diverse bio-application fields. In the workplace, the primary exposure route for TiO2 nanoparticles is inhalation through the respiratory system. Because TiO2 nanoparticles have different physiological properties, in terms of size and bioactivity, their toxic effects in the respiratory system must be determined. In this study, to determine the toxic effect of inhaled TiO2 nanoparticles in the lung and the underlying mechanism, we used a whole-body chamber inhalation system to expose A/J mice to TiO2 nanoparticles for 28 days. During the experiments, the inhaled TiO2 nanoparticles were characterized using a cascade impactor and transmission electron microscopy. After inhalation of the TiO2 nanoparticles, hyperplasia and inflammation were observed in a TiO2 dose-dependent manner. To determine the biological mechanism of the toxic response in the lung, we examined endoplasmic reticulum (ER) and mitochondria in lung. The ER and mitochondria were disrupted and dysfunctional in the TiO2-exposed lung leading to abnormal autophagy. In summary, we assessed the potential risk of TiO2 nanoparticles in the respiratory system, which contributed to our understanding of the mechanism underlining TiO2 nanoparticle toxicity in the lung.

  15. Recombinant Wheat Endoplasmic Reticulum Oxidoreductin 1 Improved Wheat Dough Properties and Bread Quality.

    PubMed

    Liu, Guang; Wang, JingJing; Hou, Yi; Huang, Yan-Bo; Zhang, Ya-Ping; Li, Cunzhi; Li, Lin; Hu, Song-Qing

    2017-03-15

    Recombinant wheat endoplasmic reticulum oxidoreductin 1 (wEro1) with considerable ability was expressed in Escherichia coli. The functional roles of wEro1 in flour processing quality were investigated by farinographic, rheological, texture profile analysis, electrophoresis, size exclusion chromatography, scanning electron microscopy, and Fourier transform infrared spectroscopy. wEro1 exhibited an obvious oxidation activity of sulfhydryl groups in small molecule and protein. Addition of wEro1 could strengthen the processing quality of dough, indicated by the improved mixing characteristics, viscoelastic properties, and bread qualities. These improvement effects of wEro1 could be attributed to the formation of macromolecular gluten polymers and massive gluten networks by disulfide cross-linking. Additionally, the increased β-turn structure further demonstrated the enhancement of dough strength. Moreover, the amount of peroxide in dough was improved significantly from 2.36 to 2.82 μmol/g of flour with 0.15% wEro1 treatment. Therefore, the results suggested that wEro1 is a promising novel flour improver.

  16. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins.

    PubMed

    Crowder, Justin J; Geigges, Marco; Gibson, Ryan T; Fults, Eric S; Buchanan, Bryce W; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G; Rubenstein, Eric M

    2015-07-24

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3'-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.

  17. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins*

    PubMed Central

    Crowder, Justin J.; Geigges, Marco; Gibson, Ryan T.; Fults, Eric S.; Buchanan, Bryce W.; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G.; Rubenstein, Eric M.

    2015-01-01

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane. PMID:26055716

  18. Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum

    PubMed Central

    Waldeck-Weiermair, Markus; Bischof, Helmut; Blass, Sandra; Deak, Andras T.; Klec, Christiane; Graier, Thomas; Roller, Clara; Rost, Rene; Eroglu, Emrah; Gottschalk, Benjamin; Hofmann, Nicole A.; Graier, Wolfgang F.; Malli, Roland

    2015-01-01

    Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store. PMID:26053751

  19. M1 of Murine Gamma-Herpesvirus 68 Induces Endoplasmic Reticulum Chaperone Production

    PubMed Central

    Feng, Jiaying; Gong, Danyang; Fu, Xudong; Wu, Ting-ting; Wang, Jane; Chang, Jennifer; Zhou, Jingting; Lu, Gang; Wang, Yibin; Sun, Ren

    2015-01-01

    Viruses rely on host chaperone network to support their infection. In particular, the endoplasmic reticulum (ER) resident chaperones play key roles in synthesizing and processing viral proteins. Influx of a large amount of foreign proteins exhausts the folding capacity in ER and triggers the unfolded protein response (UPR). A fully-executed UPR comprises signaling pathways that induce ER folding chaperones, increase protein degradation, block new protein synthesis and may eventually activate apoptosis, presenting both opportunities and threats to the virus. Here, we define a role of the MHV-68M1 gene in differential modulation of UPR pathways to enhance ER chaperone production. Ectopic expression of M1 markedly induces ER chaperone genes and expansion of ER. The M1 protein accumulates in ER during infection and this localization is indispensable for its function, suggesting M1 acts from the ER. We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK). We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production. This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways. PMID:26615759

  20. Erlotinib promotes endoplasmic reticulum stress-mediated injury in the intestinal epithelium

    SciTech Connect

    Fan, Lu; Hu, Lingna; Yang, Baofang; Fang, Xianying; Gao, Zhe; Li, Wanshuai; Sun, Yang; Shen, Yan; Wu, Xuefeng; Shu, Yongqian; Gu, Yanhong; Wu, Xudong; Xu, Qiang

    2014-07-01

    Erlotinib, a popular drug for treating non-small cell lung cancer (NSCLC), causes diarrhea in approximately 55% of patients receiving this drug. In the present study, we found that erlotinib induced barrier dysfunction in rat small intestine epithelial cells (IEC-6) by increasing epithelial permeability and down-regulating E-cadherin. The mRNA levels of various pro-inflammatory cytokines (Il-6, Il-25 and Il-17f) were increased after erlotinib treatment in IEC-6 cells. Erlotinib concentration- and time-dependently induced apoptosis and endoplasmic reticulum (ER) stress in both IEC-6 and human colon epithelial cells (CCD 841 CoN). Intestinal epithelial injury was also observed in male C57BL/6J mice administrated with erlotinib. Knockdown of C/EBP homologous protein (CHOP) with small interference RNA partially reversed erlotinib-induced apoptosis, production of IL-6 and down-regulation of E-cadherin in cultured intestinal epithelial cells. In conclusion, erlotinib caused ER stress-mediated injury in the intestinal epithelium, contributing to its side effects of diarrhea in patients. - Highlights: • Erlotinib destroyed barrier integrity both in vitro and in vivo. • Erlotinib induced inflammation both in vitro and in vivo. • Erlotinib induced apoptosis both in vitro and in vivo. • ER stress contributed to erlotinib-induced barrier dysfunction.

  1. A novel cellular stress response characterised by a rapid reorganisation of membranes of the endoplasmic reticulum

    PubMed Central

    Varadarajan, S; Bampton, E T W; Smalley, J L; Tanaka, K; Caves, R E; Butterworth, M; Wei, J; Pellecchia, M; Mitcheson, J; Gant, T W; Dinsdale, D; Cohen, G M

    2012-01-01

    Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER. PMID:22955944

  2. A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

    PubMed

    Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

    2008-11-01

    The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

  3. Emergence of endoplasmic reticulum stress and activated microglia in Purkinje cell degeneration mice.

    PubMed

    Kyuhou, Shin-ichi; Kato, Nobuo; Gemba, Hisae

    2006-03-27

    In the current studies, we characterized the molecular and cellular mechanism of cell death in Purkinje cell degeneration (pcd) mice using real-time quantitative PCR, immunohistochemistry, and Western blotting. It appears that endoplasmic reticulum (ER) stress is involved in this degeneration of Purkinje cells because ER stress-related substrates, such as CHOP and caspase 12, were strongly activated in Purkinje cells of pcd mice during the third postnatal (P) week. A significant increase in the expression of the ER-specific chaperone BiP suggested that unfolded protein responses were induced. We also found that Purkinje cells underwent apoptosis via the activation of caspase 3 and subsequent fragmentation of DNA. In addition to the activation of apoptosis in Purkinje cells, many activated microglial cells are found to be present in the molecular layer of the cerebellar cortex. In the later phase of degeneration, there was conspicuous expression of inducible nitric oxide synthase (iNOS), and some Purkinje cells were strongly labeled with an antibody to nitrotyrosine, suggesting that Purkinje cells in pcd mice are damaged by nitric oxide released from microglial cells. Administration of minocycline, which may inhibit iNOS expression, delayed the death of Purkinje cells in pcd mice and mildly improved their motor abilities. These findings suggest that ER stress participates in the degeneration of Purkinje cells and that activation of microglia accelerates Purkinje cell death in pcd mice.

  4. Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria

    PubMed Central

    Mohammed-Ali, Zahraa; Lu, Chao; Marway, Mandeep K.; Carlisle, Rachel E.; Ask, Kjetil; Lukic, Dusan; Krepinsky, Joan C.; Dickhout, Jeffrey G.

    2017-01-01

    Endoplasmic reticulum (ER) stress is implicated in chronic kidney disease (CKD) development in patients and in animal models. Here we show that ER stress inhibition through 4-phenylbutyric acid (4-PBA) administration decreases blood pressure, albuminuria, and tubular casts in an angiotensin II/deoxycorticosterone acetate/salt murine model of CKD. Lower albuminuria in 4-PBA-treated mice was associated with higher levels of cubilin protein in renal tissue membrane fractions. 4-PBA decreased renal interstitial fibrosis, renal CD3+ T-cell and macrophage infiltration, mRNA expression of TGFβ1, Wnt signaling molecules, and ER stress-induced pro-inflammatory genes. CHOP deficient mice that underwent this model of CKD developed hypertension comparable to wild type mice, but had less albuminuria and tubular casts. CHOP deficiency resulted in higher nephrin levels and decreased glomerulosclerosis compared to wild type mice; this effect was accompanied by lower macrophage infiltration and fibrosis. Our findings portray ER stress inhibition as a means to alleviate hypertensive CKD by preserving glomerular barrier integrity and tubular function. These results demonstrate ER stress modulation as a novel target for preserving renal function in hypertensive CKD. PMID:28148966

  5. Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

    PubMed

    Zito, Ester; Hansen, Henning Gram; Yeo, Giles S H; Fujii, Junichi; Ron, David

    2012-10-12

    Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

  6. Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells

    PubMed Central

    Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

    2015-01-01

    Background This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. Material/Methods HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. Results The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. Conclusions TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway. PMID:26551326

  7. Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation*

    PubMed Central

    Oh, Jisu; Riek, Amy E.; Weng, Sherry; Petty, Marvin; Kim, David; Colonna, Marco; Cella, Marina; Bernal-Mizrachi, Carlos

    2012-01-01

    Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis. PMID:22356914

  8. Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model

    PubMed Central

    Chang, Yoke-Chen; Wang, James D.; Svoboda, Kathy K.; Casillas, Robert P.; Laskin, Jeffrey D.; Gordon, Marion K.; Gerecke, Donald R.

    2013-01-01

    The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing from 24 h to 168 h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72 h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24 h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury. PMID:23357548

  9. Zonisamide suppresses endoplasmic reticulum stress-induced neuronal cell damage in vitro and in vivo.

    PubMed

    Tsujii, Saori; Ishisaka, Mitsue; Shimazawa, Masamitsu; Hashizume, Takanori; Hara, Hideaki

    2015-01-05

    Zonisamide has been reported to have protective effects on epilepsy and Parkinson׳s disease and to work via various mechanisms of action, such as inhibition of monoamine oxidase-B and enhancement of tyrosine hydroxylase. Recently, it has been suggested that zonisamide itself shows neuroprotective actions. Therefore, in the present study we investigated the neuroprotective effects of zonisamide against endoplasmic reticulum (ER) stress. We used human neuroblastoma (SH-SY5Y) cells and investigated the protective effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. In addition, we investigated the effect of zonisamide against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death and the mechanism of protection against ER stress. In vivo, we investigated the effect of zonisamide (20 mg/kg, p.o.) in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson׳s disease. Zonisamide not only suppressed MPP⁺-induced cell death, but also inhibited ER stress-induced cell death and suppressed the expression of ER stress-related factors such as C/EBO homologous protein (CHOP) in vivo. Furthermore, zonisamide inhibited the activation of caspase-3 in vitro. These results suggest that zonisamide affected ER stress via caspase-3. We think that ER stress, particularly the mechanism via caspase-3, is involved in part of the neuroprotective effect of zonisamide against the experimental models of Parkinson׳s disease.

  10. Endoplasmic reticulum stress in the peripheral nervous system is a significant driver of neuropathic pain.

    PubMed

    Inceoglu, Bora; Bettaieb, Ahmed; Trindade da Silva, Carlos A; Lee, Kin Sing Stephen; Haj, Fawaz G; Hammock, Bruce D

    2015-07-21

    Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.

  11. Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson's disease.

    PubMed

    Arduíno, Daniela Moniz; Esteves, A Raquel; Cardoso, Sandra M; Oliveira, Catarina R

    2009-09-01

    Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

  12. Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum.

    PubMed

    Moriconi, Claudia; Ordoñez, Adriana; Lupo, Giuseppe; Gooptu, Bibek; Irving, James A; Noto, Rosina; Martorana, Vincenzo; Manno, Mauro; Timpano, Valentina; Guadagno, Noemi A; Dalton, Lucy; Marciniak, Stefan J; Lomas, David A; Miranda, Elena

    2015-12-01

    The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.

  13. Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants.

    PubMed

    Liu, Jian-Xiang; Howell, Stephen H

    2010-09-01

    The endoplasmic reticulum (ER) has a sophisticated quality control (QC) system to eliminate improperly folded proteins from the secretory pathway. Given that protein folding is such a fastidious process and subject to adverse environmental conditions, the ER QC system appears to have been usurped to serve as an environmental sensor and responder in plants. Under stressful conditions, the ER protein folding machinery reaches a limit as the demands for protein folding exceed the capacity of the system. Under these conditions, misfolded or unfolded proteins accumulate in the ER, triggering an unfolded protein response (UPR). UPR mitigates ER stress by upregulating the expression of genes encoding components of the protein folding machinery or the ER-associated degradation system. In Arabidopsis thaliana, ER stress is sensed and stress signals are transduced by membrane-bound transcription factors, which are activated and mobilized under environmental stress conditions. Under acute or chronic stress conditions, UPR can also lead to apoptosis or programmed cell death. Despite recent progress in our understanding of plant protein QC, discovering how different environmental conditions are perceived is one of the major challenges in understanding this system. Since the ER QC system is one among many stress response systems in plants, another major challenge is determining the extent to which the ER QC system contributes to various stress responses in plants.

  14. Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth

    PubMed Central

    Watanabe, Keisuke; Bizen, Norihisa; Sato, Noboru; Takebayashi, Hirohide

    2016-01-01

    The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development. PMID:27959946

  15. Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum.

    PubMed

    Tabata, Keisuke; Arimoto, Masaru; Arakawa, Masashi; Nara, Atsuki; Saito, Kazunobu; Omori, Hiroko; Arai, Arisa; Ishikawa, Tomohiro; Konishi, Eiji; Suzuki, Ryosuke; Matsuura, Yoshiharu; Morita, Eiji

    2016-08-30

    Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.

  16. Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells

    PubMed Central

    Caballano-Infantes, Estefania; Terron-Bautista, José; Beltrán-Povea, Amparo; Cahuana, Gladys M; Soria, Bernat; Nabil, Hajji; Bedoya, Francisco J; Tejedo, Juan R

    2017-01-01

    Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies. PMID:28289506

  17. Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

    PubMed

    Lee, Ji Eun; Oney, McKenna; Frizzell, Kimberly; Phadnis, Nitin; Hollien, Julie

    2015-02-13

    Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

  18. Disturbed phospholipid homeostasis in endoplasmic reticulum initiates tri-o-cresyl phosphate-induced delayed neurotoxicity

    PubMed Central

    Zhu, Li; Wang, Pan; Sun, Ying-Jian; Xu, Ming-Yuan; Wu, Yi-Jun

    2016-01-01

    Tri-o-cresyl phosphate (TOCP) is a widely used organophosphorus compound, which can cause a neurodegenerative disorder, i.e., organophosphate-induced delayed neurotoxicity (OPIDN). The biochemical events in the initiation of OPIDN were not fully understood except for the essential inhibition of neuropathy target esterase (NTE). NTE, located in endoplasmic reticulum (ER), catalyzes the deacylation of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) to glycerophosphocholine (GPC). The present study aims to study the changes of ER phospholipids profile as well as levels of important intermediates of phospholipid synthesis such as diacylglycerol (DAG) and phosphatidic acid (PA) at the initiation stage of OPIDN. Hens are the most commonly used animal models of OPIDN. The spinal cord phospholipidomic profiles of hens treated by TOCP were studied by using HPLC-MS-MS. The results revealed that TOCP induced an increase of PC, LPC, and sphingomyelin (SM) levels and a decrease of GPC, phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), phosphatidylglycerol (PG), and phosphatidylinositol (PI) levels., Levels of DAG and PA were also decreased. Pretreatment with phenylmethylsulfonyl fluoride (PMSF) 24 h before TOCP administration prevented OPIDN and restored the TOCP-induced changes of phospholipids except GPC. Thus, the disruption of ER phospholipid homeostasis may contribute to the initiation of organophosphate-induced delayed neurotoxicity. PMID:27883027

  19. The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance

    PubMed Central

    Liu, Lijing; Cui, Feng; Li, Qingliang; Yin, Bojiao; Zhang, Huawei; Lin, Baoying; Wu, Yaorong; Xia, Ran; Tang, Sanyuan; Xie, Qi

    2011-01-01

    Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca2+ release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway. PMID:21187857

  20. Visualization of endoplasmic reticulum stressed cells for forward genetic studies in plants.

    PubMed

    Hayashi, Shimpei; Takaiwa, Fumio

    2015-05-15

    In eukaryotes, various cellular events are attended by a risk of triggering stress in the endoplasmic reticulum (ER). Such risks are assumed to be minimized by sophisticated regulation systems, but the nature of these systems remains largely unknown in plants. Here, transgenic Arabidopsis plants, intended for use in forward genetic studies of plant ER stress, are described. AtBiP3 promoter activity clearly reflected the effects of inducers of ER stress, such as tunicamycin, dithiothreitol, and salicylic acid. Thus transgenic plants, containing the AtBiP3 promoter coupled to a fluorescent protein-encoding gene, were generated to enable visual detection of cells experiencing ER stress in living plants. Mutagenization of these transgenic plants produced seedlings which exhibited altered fluorescence patterns. Constitutive fluorescence was observed in a number of independent lines, suggesting the plant genome includes many genes whose mutation results in ER stress. Some mutants showed strong fluorescence with different tissue specificity, implying potential ER stresses in individual cellular events. These results indicate that forward genetic approaches will provide useful information in the understanding of ER stress.

  1. Enhanced salt tolerance in tomato plants constitutively expressing heat-shock protein in the endoplasmic reticulum.

    PubMed

    Fu, C; Liu, X X; Yang, W W; Zhao, C M; Liu, J

    2016-07-14

    The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR) signaling pathway. The UPR signaling pathway is associated with plant responses to adverse environmental conditions. Thus, changes in the UPR signaling pathway might affect plant abiotic tolerance. Here, the role of ER small heat-shock protein (ER-sHSP) in improving plant resistance to salt stress was explored. Under salt stress conditions, ER-sHSP transgenic plants were found to have more vigorous roots, maintain a higher relative water content, absorb less Na(+), accumulate more osmolytes and Ca(2+), and sustain less damage to the photosystem, compared to wild-type non-transgenic plants. Furthermore, we found that the constitutive expression of ER-sHSP under salt stress depressed the expression of other ER molecular chaperones. These results indicate that the constitutive expression of ER-sHSP enhanced salinity tolerance of tomato plants significantly, and alleviated the ER stress caused by the salt stress in plant cells.

  2. Patenting the gene-hubs of endoplasmic reticulum stress: the systems biology approach.

    PubMed

    Rangel-Aldao, Rafael

    2007-01-01

    An unprecedented and accelerated process of privatizing biological information is emerging from the new techniques of systems biology as they are used to develop novel treatments to key multigenic ailments that account for a large share of mortality world-wide, including cardiovascular disease, cancer, obesity, and diabetes. The systems approach potentially allows the capture of proprietary knowledge at the cross-roads of the flow of biological information preceding these diseases, namely, from the endoplasmic reticulum stress response downstream to inflammation and disease. Although it still holds true that such pathways cannot be patented, methods and chemical substances discussed here are the subject of patents and applications by major research universities and biopharmaceutical companies to a considerable degree of overlapping information. Because biological information pathways are organized into hierarchical networks, the race seems to be on the regulation of the upstream functional modules, and because these complex networks are dominated by gene-hubs and their translation products, the winner will be the one that can appropriate specific and well described methods and substances to control the upper levels of regulation of the entire system. The road to success, however, lays formidable obstacles ahead due to the long and difficult processes separating applications being filed, from patents already issued and these from those that have survived validity litigation. It will be expected that for the sake of mankind, patents pools will be offered to develop novel therapeutics based on the biological information controlled by gene-hubs.

  3. GABAB receptor cell surface export is controlled by an endoplasmic reticulum gatekeeper

    PubMed Central

    Doly, Stéphane; Shirvani, Hamasseh; Gäta, Gabriel; Meye, Frank; Emerit, Michel-Boris; Enslen, Hervé; Achour, Lamia; Pardo-Lopez, Liliana; Kwon, Yang Seung; Armand, Vincent; Gardette, Robert; Giros, Bruno; Gassmann, Martin; Bettler, Bernhard; Mameli, Manuel; Darmon, Michèle; Marullo, Stefano

    2016-01-01

    Summary Endoplasmic reticulum (ER) release and cell surface export of many G protein-coupled receptors (GPCRs), are tightly regulated. For GABAB receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gate-keepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function. PMID:26033241

  4. Mapping intracellular diffusion distribution using single quantum dot tracking: compartmentalized diffusion defined by endoplasmic reticulum.

    PubMed

    Li, Hui; Dou, Shuo-Xing; Liu, Yu-Ru; Li, Wei; Xie, Ping; Wang, Wei-Chi; Wang, Peng-Ye

    2015-01-14

    The crowded intracellular environment influences the diffusion-mediated cellular processes, such as metabolism, signaling, and transport. The hindered diffusion of macromolecules in heterogeneous cytoplasm has been studied over years, but the detailed diffusion distribution and its origin still remain unclear. Here, we introduce a novel method to map rapidly the diffusion distribution in single cells based on single-particle tracking (SPT) of quantum dots (QDs). The diffusion map reveals the heterogeneous intracellular environment and, more importantly, an unreported compartmentalization of QD diffusions in cytoplasm. Simultaneous observations of QD motion and green fluorescent protein-tagged endoplasmic reticulum (ER) dynamics provide direct evidence that the compartmentalization results from micron-scale domains defined by ER tubules, and ER cisternae form perinuclear areas that restrict QDs to enter. The same phenomenon was observed using fluorescein isothiocyanate-dextrans, further confirming the compartmentalized diffusion. These results shed new light on the diffusive movements of macromolecules in the cell, and the mapping of intracellular diffusion distribution may be used to develop strategies for nanoparticle-based drug deliveries and therapeutics.

  5. Mitochondrial cholesterol accumulation in alcoholic liver disease: Role of ASMase and endoplasmic reticulum stress.

    PubMed

    Marí, Montserrat; Morales, Albert; Colell, Anna; García-Ruiz, Carmen; Fernández-Checa, Jose C

    2014-01-01

    Alcoholic liver disease (ALD) is a major cause of chronic liver disease and a growing health concern in theworld. While the pathogenesis of ALD is poorly characterized key players identified in experimental models and patients, such as perturbations in mitochondrial structure and function, selective loss of antioxidant defense and susceptibility to inflammatory cytokines, contribute to ALD progression. Both oxidative stress and mitochondrial dysfunction compromise essential cellular functions and energy generation and hence are important pathogenic mechanisms of ALD. An important process mediating the mitochondrial disruption induced by alcohol intake is the trafficking of cholesterol to mitochondria, mediated by acid sphingomyelinase-induced endoplasmic reticulum stress, which contributes to increased cholesterol synthesis and StARD1upregulation. Mitochondrial cholesterol accumulation not only sensitizes to oxidative stress but it can contribute to the metabolic reprogramming in ALD, manifested by activation of the hypoxia inducible transcription factor 1 and stimulation of glycolysis and lactate secretion. Thus, a better understanding of the mechanisms underlying alcohol-mediated mitochondrial impairment and oxidative stress may lead to the identification of novel treatments for ALD. The present review briefly summarizes current knowledge on the cellular and molecular mechanisms contributing to alcohol-induced mitochondrial dysfunction and cholesterol accumulation and provides insights for potential therapeutic targets in ALD.

  6. Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation

    PubMed Central

    Obata, Yuuki; Toyoshima, Shota; Wakamatsu, Ei; Suzuki, Shunichi; Ogawa, Shuhei; Esumi, Hiroyasu; Abe, Ryo

    2014-01-01

    Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation. PMID:25493654

  7. Endoplasmic reticulum stress regulates inflammation and insulin resistance in skeletal muscle from pregnant women.

    PubMed

    Liong, Stella; Lappas, Martha

    2016-04-15

    Sterile inflammation and infection are key mediators of inflammation and peripheral insulin resistance associated with gestational diabetes mellitus (GDM). Studies have shown endoplasmic reticulum (ER) stress to induce inflammation and insulin resistance associated with obesity and type 2 diabetes, however is paucity of studies investigating the effects of ER stress in skeletal muscle on inflammation and insulin resistance associated with GDM. ER stress proteins IRE1α, GRP78 and XBP-1s were upregulated in skeletal muscle of obese pregnant women, whereas IRE1α was increased in GDM women. Suppression of ER stress, using ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or siRNA knockdown of IRE1α and GRP78, significantly downregulated LPS-, poly(I:C)- or IL-1β-induced production of IL-6, IL-8, IL-1β and MCP-1. Furthermore, LPS-, poly(I:C)- or TNF-α-induced insulin resistance was improved following suppression of ER stress, by increasing insulin-stimulated phosphorylation of IR-β, IRS-1, GLUT-4 expression and glucose uptake. In summary, our inducible obesity and GDM-like models suggests that the development of GDM may be involved in activating ER stress-induced inflammation and insulin resistance in human skeletal muscle.

  8. Black tea protects against hypertension-associated endothelial dysfunction through alleviation of endoplasmic reticulum stress

    PubMed Central

    San Cheang, Wai; Yuen Ngai, Ching; Yen Tam, Ye; Yu Tian, Xiao; Tak Wong, Wing; Zhang, Yang; Wai Lau, Chi; Chen, Zhen Yu; Bian, Zhao-Xiang; Huang, Yu; Ping Leung, Fung

    2015-01-01

    Hypertensive patients have been found to be associated with elevated levels of homocysteine, known as hyperhomocysteinemia. Homocysteine (Hcy) can induce endoplasmic reticulum (ER) stress in endothelial cells. This study aims to investigate whether black tea (BT) protects against hypertension-associated endothelial dysfunction through alleviation of ER stress. Rat aortae and cultured rat aortic endothelial cells were treated with Hcy, BT extract, and theaflavin-3,3’-digallate (TF3). Male Sprague Dawley rats were infused with angiotensin II (Ang II) to induce hypertension and orally administrated with BT extract at 15 mg/kg/day for 2 weeks. Hcy impaired endothelium-dependent relaxations of rat aortae and led to ER stress in endothelial cells, which were ameliorated by co-incubation of BT extract and TF3. The blood pressure of Ang II-infused rats and plasma Hcy level were normalized by BT consumption. Impaired endothelium-dependent relaxations in renal arteries, carotid arteries and aortae, and flow-mediated dilatations in third-order mesenteric resistance arteries were improved. Elevations of ER stress markers and ROS level, plus down-regulation of Hcy metabolic enzymes in aortae from Ang II-infused rats were prevented by BT treatment. Our data reveal the novel cardiovascular benefits of BT in ameliorating vascular dysfunctions, providing insight into developing BT into beneficial dietary supplements in hypertensive patients. PMID:25976123

  9. Disassembly of Simian Virus 40 during Passage through the Endoplasmic Reticulum and in the Cytoplasm

    PubMed Central

    Kuksin, Dmitry

    2012-01-01

    The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection. PMID:22090139

  10. Disassembly of simian virus 40 during passage through the endoplasmic reticulum and in the cytoplasm.

    PubMed

    Kuksin, Dmitry; Norkin, Leonard C

    2012-02-01

    The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection.

  11. XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death

    PubMed Central

    Oishi, N; Duscha, S; Boukari, H; Meyer, M; Xie, J; Wei, G; Schrepfer, T; Roschitzki, B; Boettger, E C; Schacht, J

    2015-01-01

    Here we study links between aminoglycoside-induced mistranslation, protein misfolding and neuropathy. We demonstrate that aminoglycosides induce misreading in mammalian cells and assess endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways. Genome-wide transcriptome and proteome analyses revealed upregulation of genes related to protein folding and degradation. Quantitative PCR confirmed induction of UPR markers including C/EBP homologous protein, glucose-regulated protein 94, binding immunoglobulin protein and X-box binding protein-1 (XBP1) mRNA splicing, which is crucial for UPR activation. We studied the effect of a compromised UPR on aminoglycoside ototoxicity in haploinsufficient XBP1 (XBP1+/−) mice. Intra-tympanic aminoglycoside treatment caused high-frequency hearing loss in XBP1+/− mice but not in wild-type littermates. Densities of spiral ganglion cells and synaptic ribbons were decreased in gentamicin-treated XBP1+/− mice, while sensory cells were preserved. Co-injection of the chemical chaperone tauroursodeoxycholic acid attenuated hearing loss. These results suggest that aminoglycoside-induced ER stress and cell death in spiral ganglion neurons is mitigated by XBP1, masking aminoglycoside neurotoxicity at the organismal level. PMID:25973683

  12. Chlamydiae Assemble a Pathogen Synapse to Hijack the Host Endoplasmic Reticulum

    PubMed Central

    Dumoux, Maud; Clare, Daniel K; Saibil, Helen R; Hayward, Richard D

    2012-01-01

    Chlamydiae are obligate intracellular bacterial pathogens that replicate within a specialized membrane-bound compartment, termed an ‘inclusion’. The inclusion membrane is a critical host–pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. Here we show that the host endoplasmic reticulum (ER) is specifically recruited to the inclusion, and that key rough ER (rER) proteins are enriched on and translocated into the inclusion. rER recruitment is a Chlamydia-orchestrated process that occurs independently of host trafficking. Generation of infectious progeny requires an intact ER, since ER vacuolation early during infection stalls inclusion development, whereas disruption post ER recruitment bursts the inclusion. Electron tomography and immunolabelling of Chlamydia-infected cells reveal ‘pathogen synapses’ at which ordered arrays of chlamydial type III secretion complexes connect to the inclusion membrane only at rER contact sites. Our data show a supramolecular assembly involved in pathogen hijack of a key host organelle. PMID:22901061

  13. Endoplasmic reticulum stress as a novel cellular response to di (2-ethylhexyl) phthalate exposure.

    PubMed

    Peropadre, Ana; Fernández Freire, Paloma; Pérez Martín, José Manuel; Herrero, Óscar; Hazen, María José

    2015-12-25

    Di (2-ethylhexyl) phthalate is a high-production chemical widely used as a plasticizer for polyvinyl chloride products. Due to its ubiquitous presence in environmental compartments and the constant exposure of the general population through ingestion, inhalation, and dermal absorption, this compound has been subjected to extensive in vivo and in vitro toxicological studies. Despite the available information, research on the cytotoxicity of di (2-ethylhexyl) phthalate in mammalian cells is relatively limited.In this paper, an in vitro multi-parametric approach was used to provide further mechanistic data on the toxic activity of this chemical in Vero and HaCaT cells. Our results reveal that a 24 h exposure to di (2-ethylhexyl) phthalate causes, in both cell lines, an inhibition of cell proliferation that was linked to cell cycle delay at the G1 phase. Concomitantly, the tested compound induces mild endoplasmic reticulum stress which leads to an adaptive rather than a pro-apoptotic response in mammalian cells. These findings demonstrate that there are multiple potential cellular targets of di (2-ethylhexyl) phthalate-induced toxicity and the need to develop further experimental studies for the risk assessment of this ubiquitous plasticizer.

  14. KUS121, a VCP modulator, attenuates ischemic retinal cell death via suppressing endoplasmic reticulum stress

    PubMed Central

    Hata, Masayuki; Ikeda, Hanako O.; Kikkawa, Chinami; Iwai, Sachiko; Muraoka, Yuki; Hasegawa, Tomoko; Kakizuka, Akira; Yoshimura, Nagahisa

    2017-01-01

    Ischemic neural damages cause several devastating diseases, including brain stroke and ischemic retinopathies, and endoplasmic reticulum (ER) stress has been proposed to be the underlying mechanism of the neuronal cell death of these conditions. We previously synthesized Kyoto University substances (KUSs) as modulators of valosin-containing protein (VCP); KUSs inhibit VCP ATPase activity and protect cells from different cell death-inducing insults. Here, we examined the efficacy of KUS121 in a rat model of retinal ischemic injury. Systemic administration of KUS121 to rats with ischemic retinal injury significantly suppressed inner retinal thinning and death of retinal ganglion and amacrine cells, with a significant functional maintenance of visual functions, as judged by electroretinography. Furthermore, intravitreal injection of KUS121, which is the clinically preferred route of drug administration for retinal diseases, appeared to show an equal or better neuroprotective efficacy in the ischemic retina compared with systemic administration. Indeed, induction of the ER stress marker C/EBP homologous protein (CHOP) after the ischemic insult was significantly suppressed by KUS121 administration. Our study suggests VCP modulation by KUS as a promising novel therapeutic strategy for ischemic neuronal diseases. PMID:28317920

  15. Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis.

    PubMed

    Villeneuve, Julien; Duran, Juan; Scarpa, Margherita; Bassaganyas, Laia; Van Galen, Josse; Malhotra, Vivek

    2017-01-01

    Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi-cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor-mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain-containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions.

  16. Endoplasmic Reticulum Ca2+ Handling in Excitable Cells in Health and Disease

    PubMed Central

    Mattson, Mark P.

    2011-01-01

    The endoplasmic reticulum (ER) is a morphologically and functionally diverse organelle capable of integrating multiple extracellular and internal signals and generating adaptive cellular responses. It plays fundamental roles in protein synthesis and folding and in cellular responses to metabolic and proteotoxic stress. In addition, the ER stores and releases Ca2+ in sophisticated scenarios that regulate a range of processes in excitable cells throughout the body, including muscle contraction and relaxation, endocrine regulation of metabolism, learning and memory, and cell death. One or more Ca2+ ATPases and two types of ER membrane Ca2+ channels (inositol trisphosphate and ryanodine receptors) are the major proteins involved in ER Ca2+ uptake and release, respectively. There are also direct and indirect interactions of ER Ca2+ stores with plasma membrane and mitochondrial Ca2+-regulating systems. Pharmacological agents that selectively modify ER Ca2+ release or uptake have enabled studies that revealed many different physiological roles for ER Ca2+ signaling. Several inherited diseases are caused by mutations in ER Ca2+-regulating proteins, and perturbed ER Ca2+ homeostasis is implicated in a range of acquired disorders. Preclinical investigations suggest a therapeutic potential for use of agents that target ER Ca2+ handling systems of excitable cells in disorders ranging from cardiac arrhythmias and skeletal muscle myopathies to Alzheimer disease. PMID:21737534

  17. Role of Mitochondria-Associated Endoplasmic Reticulum Membrane in Inflammation-Mediated Metabolic Diseases

    PubMed Central

    Jeon, Jae-Han

    2016-01-01

    Inflammation is considered to be one of the most critical factors involved in the development of complex metabolic diseases such as type 2 diabetes, cancer, and cardiovascular disease. A few decades ago, the discovery of mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) was followed by the identification of its roles in regulating cellular homeostatic processes, ranging from cellular bioenergetics to apoptosis. MAM provides an excellent platform for numerous signaling pathways; among them, inflammatory signaling pathways associated with MAM play a critical role in cellular defense during pathogenic infections and metabolic disorders. However, induction of MAM causes deleterious effects by amplifying mitochondrial reactive oxygen species generation through increased calcium transfer from the ER to mitochondria, thereby causing mitochondrial damage and release of mitochondrial components into the cytosol as damage-associated molecular patterns (DAMPs). These mitochondrial DAMPs rapidly activate MAM-resident inflammasome components and other inflammatory factors, which promote inflammasome complex formation and release of proinflammatory cytokines in pathological conditions. Long-term stimulation of the inflammasome instigates chronic inflammation, leading to the pathogenesis of metabolic diseases. In this review, we summarize the current understanding of MAM and its association with inflammation-mediated metabolic diseases. PMID:28074080

  18. Transport of phosphatidylserine from the endoplasmic reticulum to the site of phosphatidylserine decarboxylase2 in yeast.

    PubMed

    Kannan, Muthukumar; Riekhof, Wayne R; Voelker, Dennis R

    2015-02-01

    Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport-dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p-specific transport pathway is one in which the enzyme and its non-catalytic N-terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.

  19. Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

    PubMed Central

    Casey, Amanda K.; Chen, Shuliang; Novick, Peter; Ferro-Novick, Susan; Wente, Susan R.

    2015-01-01

    The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions. PMID:26041935

  20. The role of endoplasmic reticulum stress and insulin resistance in the occurrence of goose fatty liver.

    PubMed

    Geng, Tuoyu; Xia, Lili; Li, Fuyuan; Xia, Jing; Zhang, Yihui; Wang, Qianqian; Yang, Biao; Montgomery, Sean; Cui, Hengmi; Gong, Daoqing

    2015-09-11

    In mammals, insulin resistance (IR) is required for the occurrence of non-alcoholic fatty liver disease, and endoplasmic reticulum stress (ERS) contributes to IR. As geese have physiological and metabolic characteristics different from mammals, it is unclear whether these mechanisms also underlie the occurrence of goose fatty liver. To address this, 70-day-old geese were treated with an ERS inducer or overfed, and variables associated with ERS or IR were subsequently determined. The data indicated that the group of geese treated with the ERS inducer for 20d appeared to be more intolerant to blood glucose than the control group, and their livers showed features of hepatic steatosis, suggesting ERS can induce IR and hepatic steatosis in geese. In contrast, overfeeding did not induce ERS, probably due to the upregulated expression of fatty acid desaturases, but induced higher fasting/postprandial blood glucose as well as glucose intolerance in geese, which was accompanied by a dramatic increase of liver weight. Taken together, these findings delineated the role of ERS and IR in the occurrence of goose fatty liver.

  1. Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

    PubMed Central

    Ishii, Kiyoaki; Hirose, Kenzo; Iino, Masamitsu

    2006-01-01

    Although many cell functions are regulated by Ca2+ oscillations induced by a cyclic release of Ca2+ from intracellular Ca2+ stores, the pacemaker mechanism of Ca2+ oscillations remains to be explained. Using green fluorescent protein-based Ca2+ indicators that are targeted to intracellular Ca2+ stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca2+ shuttles between the ER and mitochondria in phase with Ca2+ oscillations. Following agonist stimulation, Ca2+ release from the ER generated the first Ca2+ oscillation and loaded mitochondria with Ca2+. Before the second Ca2+ oscillation, Ca2+ release from the mitochondria by means of the Na+/Ca2+ exchanger caused a gradual increase in cytoplasmic Ca2+ concentration, inducing a regenerative ER Ca2+ release, which generated the peak of Ca2+ oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca2+ was depleted. Thus, Ca2+ shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca2+ oscillations. PMID:16415789

  2. Hyperglycemia magnifies bupivacaine-induced cell apoptosis triggered by mitochondria dysfunction and endoplasmic reticulum stress.

    PubMed

    Li, Le; Ye, Xiao-ping; Lu, Ai-zhu; Zhou, Shu-qin; Liu, Hui; Liu, Zhong-jie; Jiang, Shan; Xu, Shi-yuan

    2013-06-01

    Nerve cell injury associated with apoptosis plays an important role in the development of diabetic peripheral neuropathy (DPN). However, it remains unclear whether preexisting or potential neurocyte damage induced by hyperglycemia increases sensitivity to local anesthetics. SH-SY5Y cells were pretreated with a high concentration of glucose in vitro, to imitate DPN prior to administration of bupivacaine or placebo. Cell viability and apoptosis were investigated with a CCK-8 assay and flow cytometry, respectively. In addition, mitochondrial membrane potential, reactive oxygen species (ROS), mitochondrially generated ROS, and activity of mitochondrial complexes I and III were studied to explore the molecular mechanism of bupivacaine-induced mitochondrial injury. Grp78 and caspase-12 expression were measured by qRT-PCR and Western blot, representing endoplasmic reticulum (ER) stress. Cell structure was also assessed via transmission electron microscopy. Incubation with bupivacaine decreased the activity of mitochondrial complexes I and III; increased ROS production at cell and mitochondrial levels, mitochondrial potential depolarization, and Grp78 and caspase-12 expression at both transcription and translation levels; and affected cell structure, which could be enhanced by glucose pretreatment. These findings indicate that mitochondrial dysfunction and ER stress related to ROS are involved in bupivacaine-induced apoptosis and may be enhanced by glucose administration.

  3. Endoplasmic Reticulum Stress and Autophagy in Homocystinuria Patients with Remethylation Defects

    PubMed Central

    Martínez-Pizarro, Ainhoa; Desviat, Lourdes R.; Ugarte, Magdalena; Pérez, Belén; Richard, Eva

    2016-01-01

    Proper function of endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs) have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patient-derived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IP3R1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2) were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patient-derived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca2+ homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications. PMID:26959487

  4. Quantity control of the ErbB3 receptor tyrosine kinase at the endoplasmic reticulum.

    PubMed

    Fry, William H D; Simion, Catalina; Sweeney, Colleen; Carraway, Kermit L

    2011-07-01

    The ErbB3 receptor tyrosine kinase contributes to a variety of developmental processes, and its overexpression and aberrant activation promote tumor progression and therapeutic resistance. Accumulating evidence suggests that tumor overexpression may be mediated by the loss of posttranscriptional negative regulatory mechanisms, such as protein degradation, that normally keep receptor levels in check. Our previous studies indicate that the RING finger E3 ubiquitin ligase Nrdp1, a protein lost in breast and other tumor types, suppresses ErbB3 levels by mediating ligand-independent receptor ubiquitination and degradation. Here we demonstrate that Nrdp1 preferentially associates with the nascent form of ErbB3 to accelerate its degradation, and we show that the two proteins colocalize at the endoplasmic reticulum (ER). Blocking the exit of ErbB3 from the ER does not affect the ability of Nrdp1 to mediate receptor ubiquitination or degradation, while functional disruption of the conserved ER-associated degradation (ERAD) pathway ATPase VCP/p97 leads to the Nrdp1-dependent accumulation of ubiquitinated ErbB3 but blocks receptor degradation. Further evidence indicates that the ErbB3 targeted by Nrdp1 for degradation is properly folded and fully functional. Collectively, these observations point to a novel mechanism of receptor tyrosine kinase quantity control wherein steady-state levels of signaling-competent receptor are dictated by an ER-localized degradation pathway.

  5. Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle

    PubMed Central

    Koh, Ho-Jin; Toyoda, Taro; Didesch, Michelle M.; Lee, Min-Young; Sleeman, Mark W.; Kulkarni, Rohit N.; Musi, Nicolas; Hirshman, Michael F.; Goodyear, Laurie J.

    2013-01-01

    Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle. PMID:23695665

  6. Unexpected blockade of adipocyte differentiation by K-7174: Implication for endoplasmic reticulum stress

    SciTech Connect

    Shimada, Tsuyoshi; Hiramatsu, Nobuhiko; Okamura, Maro; Hayakawa, Kunihiro; Kasai, Ayumi; Yao, Jian; Kitamura, Masanori

    2007-11-16

    Preadipocytes constitutively express GATA-2 and GATA-3 that are required to halt the cells at the undifferentiated stage. However, we unexpectedly found that K-7174, a GATA-specific inhibitor, did not induce but rather inhibited differentiation of 3T3-L1 preadipocytes. It was associated with lack of lipid accumulation, blunted expression of adipocyte markers including adiponectin and peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), and sustained expression of a preadipocyte marker monocyte chemoattractant protein 1 (MCP-1). Subsequent experiments revealed that K-7174 had the potential to induce endoplasmic reticulum (ER) stress evidenced by induction of GRP78 and CHOP. Other inducers of ER stress completely reproduced the effects of K-7174 including suppression of lipid accumulation, blockade of induction of adiponection and PPAR{gamma} and maintenance of MCP-1 expression. These results indicated a possibility that ER stress suppresses adipocyte differentiation and that GATA inhibitor K-7174 has the potential for interfering with adipogenesis through induction of ER stress.

  7. Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.

    PubMed

    Matsunaga, Kohichi; Morita, Eiji; Saitoh, Tatsuya; Akira, Shizuo; Ktistakis, Nicholas T; Izumi, Tetsuro; Noda, Takeshi; Yoshimori, Tamotsu

    2010-08-23

    Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

  8. Effects of endoplasmic reticulum stress on the expression of inflammatory cytokines in patients with ulcerative colitis

    PubMed Central

    Li, Nan; Wang, Xue-Ming; Jiang, Li-Jun; Zhang, Meng; Li, Na; Wei, Zhen-Zhen; Zheng, Nan; Zhao, Ya-Jiao

    2016-01-01

    AIM: To explore the changes of X-box binding protein 1 splicing (XBP1s) and inflammatory cytokine expression in patients with ulcerative colitis (UC) in response to endoplasmic reticulum stress (ERS). METHODS: Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction were performed to detect the forms of XBP1s and the expression of interleukin (IL)-2, interferon (IFN)-γ, and IL-17α. Differences between patients with UC and normal subjects were then determined. RESULTS: Mononuclear cells of the peripheral blood of normal subjects and UC patients with were stimulated with no drugs (control), phytohemagglutinin (PHA), thapsigargin (TG), or both PHA and TG. XBP1s in patients with UC exhibited splicing, which was greater with co-stimulation than single stimulation. Co-stimulation increased the expression level of IL-2, IFN-γ, and IL-17α. CONCLUSION: The T lymphocytes of both normal subjects and patients with UC responded to ERS by activating the XBP1s-mediated signalling pathway, upregulating the expression of inflammatory cytokines, and increasing the occurrence of inflammation. The mononuclear cells in the peripheral blood of patients with UC were more sensitive to ERS than those in the peripheral blood of normal subjects. PMID:26900298

  9. Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum Stress and Attenuates Kidney Injury.

    PubMed

    Mami, Iadh; Bouvier, Nicolas; El Karoui, Khalil; Gallazzini, Morgan; Rabant, Marion; Laurent-Puig, Pierre; Li, Shuping; Tharaux, Pierre-Louis; Beaune, Philippe; Thervet, Eric; Chevet, Eric; Hu, Guo-Fu; Pallet, Nicolas

    2016-03-01

    Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang(-/-)) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism.

  10. Contribution of mitochondria and endoplasmic reticulum dysfunction in insulin resistance: Distinct or interrelated roles?

    PubMed

    Rieusset, J

    2015-11-01

    Mitochondria and the endoplasmic reticulum (ER) regulate numerous cellular processes, and are critical contributors to cellular and whole-body homoeostasis. More important, mitochondrial dysfunction and ER stress are both closely associated with hepatic and skeletal muscle insulin resistance, thereby playing crucial roles in altered glucose homoeostasis in type 2 diabetes mellitus (T2DM). The accumulated evidence also suggests a potential interrelationship between alterations in both types of organelles, as mitochondrial dysfunction could participate in activation of the unfolded protein response, whereas ER stress could influence mitochondrial function. The fact that mitochondria and the ER are physically and functionally interconnected via mitochondria-associated membranes (MAMs) supports their interrelated roles in the pathophysiology of T2DM. However, the mechanisms that coordinate the interplay between mitochondrial dysfunction and ER stress, and its relevance to the control of glucose homoeostasis, are still unknown. This review evaluates the involvement of mitochondria and ER independently in the development of peripheral insulin resistance, as well as their potential roles in the disruption of organelle crosstalk at MAM interfaces in the alteration of insulin signalling.

  11. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  12. Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity.

    PubMed

    Arruda, Ana Paula; Pers, Benedicte M; Parlakgül, Güneş; Güney, Ekin; Inouye, Karen; Hotamisligil, Gökhan S

    2014-12-01

    Proper function of the endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site has been linked to pathophysiological states, including metabolic diseases. Although the ER and mitochondria play distinct cellular roles, these organelles also form physical interactions with each other at sites defined as mitochondria-associated ER membranes (MAMs), which are essential for calcium, lipid and metabolite exchange. Here we show that in the liver, obesity leads to a marked reorganization of MAMs resulting in mitochondrial calcium overload, compromised mitochondrial oxidative capacity and augmented oxidative stress. Experimental induction of ER-mitochondria interactions results in oxidative stress and impaired metabolic homeostasis, whereas downregulation of PACS-2 or IP3R1, proteins important for ER-mitochondria tethering or calcium transport, respectively, improves mitochondrial oxidative capacity and glucose metabolism in obese animals. These findings establish excessive ER-mitochondrial coupling as an essential component of organelle dysfunction in obesity that may contribute to the development of metabolic pathologies such as insulin resistance and diabetes.

  13. Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

    PubMed Central

    Kutay, U; Ahnert-Hilger, G; Hartmann, E; Wiedenmann, B; Rapoport, T A

    1995-01-01

    Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion. Images PMID:7835332

  14. Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress.

    PubMed

    Su, Lijuan; Chen, Xiaoyi; Wu, Jun; Lin, Biyun; Zhang, Haitao; Lan, Liubo; Luo, Hui

    2013-12-01

    Prolonged endoplasmic reticulum (ER) stress may activate apoptotic pathways in cancer cells. It is suggested that ER stress has the potential of enhancing tumor death in cancer therapy. Galangin, a flavonol derived from Alpinia officinarum Hance, has been shown to suppress the proliferation of hepatocellular carcinoma cells (HCC). The aim of this study was to determine whether galangin was able to induce ER stress in HepG2, Hep3B and PLC/PRF/5 cells. The proliferation of HCC was tested by MTT method. Intracellular Ca(2+) levels were measured with Fluo3-AM.The proteins levels of GRP94, GRP78 and CHOP were detected by Western blot. To further understand the anti-HCC mechanism of galangin, mitogen-activated protein kinases (MAPKs) were detected. The results showed that galangin treatment induced ER stress was evidenced by increased protein levels of GRP94, GRP78 and CHOP, as well as increased free cytosolic Ca(2+) concentration. ER stress inhibitor 4-PBA and CHOP siRNA blocked significantly galangin-induced ER in all three cell lines. Further experiments showed that MAPKs involved in ER stress induced by galangin. In summary, galangin is identified as a stimulator of ER stress to suppress the proliferation of HCC, and may be used as a potential anti-cancer agent.

  15. Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress.

    PubMed

    Li, Jian-Shuang; Wang, Wen-Jun; Sun, Yu; Zhang, Yu-Hao; Zheng, Ling

    2015-05-01

    Ursolic acid (UA) is a natural pentacyclic triterpenoid compound, which is enriched with many herbs and plants, such as apple, cranberry and olive. UA performs multiple biological activities including anti-oxidation, anti-inflammation, anti-cancer and hepatoprotection. However, the exact mechanism underlying the hepatoprotective activity of UA remains unclear. In this study, the effects of UA on the development of nonalcoholic fatty liver disease (NAFLD) were investigated. In vivo, UA treatment (0.14%, w/w) significantly decreased the liver weight, serum levels of ALT/AST and hepatic steatosis in db/db mice (a type 2 diabetic mouse model). In vitro, UA treatment (10-30 μg ml(-1)) significantly decreased palmitic acid induced intracellular lipid accumulation in L02 cells. Our results suggested that the beneficial effects of UA on NAFLD may be due to its ability to increase lipid β-oxidation and to inhibit the hepatic endoplasmic reticulum (ER) stress. Together, UA may be further considered as a natural compound for NAFLD treatment.

  16. HDAC Inhibitors and RECK Modulate Endoplasmic Reticulum Stress in Tumor Cells

    PubMed Central

    Chen, Yun; Tsai, Ya-Hui; Tseng, Sheng-Hong

    2017-01-01

    In the tumor microenvironment hypoxia and nutrient deprived states can induce endoplasmic reticulum (ER) stress. If ER stress is not relieved, the tumor cells may become apoptotic. Therefore, targeting ER homeostasis is a potential strategy for cancer treatment. Various chemotherapeutic agents including histone deacetylase (HDAC) inhibitors can induce ER stress to cause cell death in cancers. Some HDAC inhibitors can prevent HDAC from binding to the specificity protein 1-binding site of the promoter of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and up-regulate RECK expression. Up-regulation of RECK expression by HDAC inhibitors has been observed in various cancer types. RECK is a tumor and metastasis suppressor gene and is critical for regulating tumor cell invasiveness and metastasis. RECK also modulates ER stress via binding to and sequestering glucose-regulated protein 78 protein, so that the transmembrane sensors, such as protein kinase RNA-like ER kinase are released to activate eukaryotic translational initiation factor 2α phosphorylation and enhance ER stress. Therefore, HDAC inhibitors may directly induce ER stress or indirectly induce this stress by up-regulating RECK in cancer cells. PMID:28134767

  17. Endoplasmic reticulum-mitochondrial crosstalk: a novel role for the mitochondrial peptide humanin

    PubMed Central

    Sreekumar, Parameswaran G.; Hinton, David R.; Kannan, Ram

    2017-01-01

    In this review, the interactive mechanisms of mitochondria with the endoplasmic reticulum (ER) are discussed with emphasis on the potential protective role of the mitochondria derived peptide humanin (HN) in ER stress. The ER and mitochondria are dynamic organelles capable of modifying their structure and function in response to changing environmental conditions. The ER and mitochondria join together at multiple sites and form mitochondria-ER associated membranes that participate in signal transduction pathways that are under active investigation. Our laboratory previously showed that HN protects cells from oxidative stress induced cell death and more recently, described the beneficial role of HN on ER stress-induced apoptosis in retinal pigment epithelium cells and the involvement of ER-mitochondrial cross-talk in cellular protection. The protection was achieved, in part, by the restoration of mitochondrial glutathione that was depleted by ER stress. Thus, HN may be a promising candidate for therapy for diseases that involve both oxidative and ER stress. Developing novel approaches for retinal delivery of HN, its analogues as well as small molecular weight ER stress inhibitors would prove to be a valuable approach in the treatment of age-related macular degeneration. PMID:28250736

  18. The yeast p5 type ATPase, spf1, regulates manganese transport into the endoplasmic reticulum.

    PubMed

    Cohen, Yifat; Megyeri, Márton; Chen, Oscar C W; Condomitti, Giuseppe; Riezman, Isabelle; Loizides-Mangold, Ursula; Abdul-Sada, Alaa; Rimon, Nitzan; Riezman, Howard; Platt, Frances M; Futerman, Anthony H; Schuldiner, Maya

    2013-01-01

    The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn(2+) homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn(2+) in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+) we could observe concurrent reduction in many Mn(2+)-related process in the ER lumen. Conversely, cytosolic Mn(2+)-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+) transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn(2+)-dependent neurological disorders.

  19. A small deletion in SERPINC1 causes type I antithrombin deficiency by promoting endoplasmic reticulum stress

    PubMed Central

    Cai, Lei; Zhang, Xin; Zhai, Yu; Liu, Jianren

    2016-01-01

    Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency. PMID:27708219

  20. Methyl jasmonate affects morphology, number and activity of endoplasmic reticulum bodies in Raphanus sativus root cells.

    PubMed

    Gotté, Maxime; Ghosh, Rajgourab; Bernard, Sophie; Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Hara-Nishimura, Ikuko; Driouich, Azeddine

    2015-01-01

    The endoplasmic reticulum (ER) bodies are ER-derived structures that are found in Brassicaceae species and thought to play a role in defense. Here, we have investigated the occurrence, distribution and function of ER bodies in root cells of Raphanus sativus using a combination of microscopic and biochemical methods. We have also assessed the response of ER bodies to methyl jasmonate (MeJA), a phytohormone that mediates plant defense against wounding and pathogens. Our results show that (i) ER bodies do occur in different root cell types from the root cap region to the differentiation zone; (ii) they do accumulate a PYK10-like protein similar to the major marker protein of ER bodies that is involved in defense in Arabidopsis thaliana; and (iii) treatment of root cells with MeJA causes a significant increase in the number of ER bodies and the activity of β-glucosidases. More importantly, MeJA was found to induce the formation of very long ER bodies that results from the fusion of small ones, a phenomenon that has not been reported in any other study so far. These findings demonstrate that MeJA impacts the number and morphology of functional ER bodies and stimulates ER body enzyme activities, probably to participate in defense responses of radish root. They also suggest that these structures may provide a defensive system specific to root cells.

  1. In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

    PubMed Central

    Hendershot, L M; Wei, J Y; Gaut, J R; Lawson, B; Freiden, P J; Murti, K G

    1995-01-01

    BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo. Images PMID:7612964

  2. Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors

    PubMed Central

    Valenzuela, José I.; Jaureguiberry-Bravo, Matías; Salas, Daniela A.; Ramírez, Omar A.; Cornejo, Víctor H.; Lu, Hsiangmin E.; Blanpied, Thomas A.; Couve, Andrés

    2014-01-01

    ABSTRACT In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles. PMID:24895402

  3. Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells.

    PubMed

    Li, Tianliang; Su, Ling; Zhong, Ning; Hao, Xuexi; Zhong, Diansheng; Singhal, Sunil; Liu, Xiangguo

    2013-07-01

    Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

  4. Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum

    PubMed Central

    Verchot, Jeanmarie

    2016-01-01

    The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus–host interactions will provide new targets for crop improvement. PMID:27869775

  5. Quantitative proteomics reveal proteins enriched in tubular endoplasmic reticulum of Saccharomyces cerevisiae

    PubMed Central

    Wang, Xinbo; Li, Shanshan; Wang, Haicheng; Shui, Wenqing; Hu, Junjie

    2017-01-01

    The tubular network is a critical part of the endoplasmic reticulum (ER). The network is shaped by the reticulons and REEPs/Yop1p that generate tubules by inducing high membrane curvature, and the dynamin-like GTPases atlastin and Sey1p/RHD3 that connect tubules via membrane fusion. However, the specific functions of this ER domain are not clear. Here, we isolated tubule-based microsomes from Saccharomyces cerevisiae via classical cell fractionation and detergent-free immunoprecipitation of Flag-tagged Yop1p, which specifically localizes to ER tubules. In quantitative comparisons of tubule-derived and total microsomes, we identified a total of 79 proteins that were enriched in the ER tubules, including known proteins that organize the tubular ER network. Functional categorization of the list of proteins revealed that the tubular ER network may be involved in membrane trafficking, lipid metabolism, organelle contact, and stress sensing. We propose that affinity isolation coupled with quantitative proteomics is a useful tool for investigating ER functions. DOI: http://dx.doi.org/10.7554/eLife.23816.001 PMID:28287394

  6. Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2.

    PubMed

    Ascher, David B; Polekhina, Galina; Parker, Michael W

    2012-04-01

    Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the `molecular-ruler' mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure-function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures.

  7. Endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate.

    PubMed

    Brookes, Steven J; Barron, Martin J; Boot-Handford, Ray; Kirkham, Jennifer; Dixon, Michael J

    2014-05-01

    Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease.

  8. Rab6 is increased in Alzheimer's disease brain and correlates with endoplasmic reticulum stress.

    PubMed

    Scheper, W; Hoozemans, J J M; Hoogenraad, C C; Rozemuller, A J M; Eikelenboom, P; Baas, F

    2007-10-01

    Alzheimer's disease (AD) is characterized by deposits of aggregated proteins. Accumulation of aggregation-prone proteins activates protein quality control mechanisms, such as the unfolded protein response (UPR) in the endoplasmic reticulum (ER). We previously reported upregulation of the UPR marker BiP in AD brain. In this study, we investigated the small GTPase Rab6, which is involved in retrograde Golgi-ER trafficking and may function as a post-ER quality control system. Using immunohistochemistry and semiquantitative Western blotting, the expression of Rab6 was analysed in hippocampus, entorhinal and temporal cortex of 10 AD patients and six nondemented control subjects. Rab6 is upregulated in AD temporal cortex from Braak stage 3/4, the same stage that UPR activation is found. We observe increased neuronal Rab6 immunoreactivity in all brain areas examined. Although some neurones show colocalization of immunoreactivity for Rab6 and hyperphosphorylated tau, strong Rab6 staining does not colocalize with tangles. We find a highly significant correlation between the Rab6 and BiP levels. In vitro data show that Rab6 is not upregulated as a result of UPR activation or proteasome inhibition indicating an independent regulatory mechanism. Our data suggest that ER and post-ER protein quality control mechanisms are activated early in the pathology of AD.

  9. Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection.

    PubMed

    Grangeon, Romain; Agbeci, Maxime; Chen, Jun; Grondin, Gilles; Zheng, Huanquan; Laliberté, Jean-François

    2012-09-01

    The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.

  10. IRE1α is an endogenous substrate of endoplasmic reticulum-associated degradation

    PubMed Central

    Sun, Shengyi; Shi, Guojun; Sha, Haibo; Ji, Yewei; Han, Xuemei; Shu, Xin; Ma, Hongming; Inoue, Takamasa; Gao, Beixue; Kim, Hana; Bu, Pengcheng; Guber, Robert D.; Shen, Xiling; Lee, Ann-Hwee; Iwawaki, Takao; Paton, Adrienne W.; Paton, James C.; Fang, Deyu; Tsai, Billy; Yates, John R.; Wu, Haoquan; Kersten, Sander; Long, Qiaoming; Duhamel, Gerald E.; Simpson, Kenneth W.; Qi, Ling

    2015-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both intramembrane hydrophilic residues of IRE1α and lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and appear normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signaling in vivo by managing its protein turnover. PMID:26551274

  11. Endoplasmic reticulum-associated mitochondria-cortex tether functions in the distribution and inheritance of mitochondria.

    PubMed

    Lackner, Laura L; Ping, Holly; Graef, Martin; Murley, Andrew; Nunnari, Jodi

    2013-02-05

    To elucidate the functional roles of mitochondrial dynamics in vivo, we identified genes that become essential in cells lacking the dynamin-related proteins Fzo1 and Dnm1, which are required for mitochondrial fusion and division, respectively. The screen identified Num1, a cortical protein implicated in mitochondrial division and distribution that also functions in nuclear migration. Our data indicate that Num1, together with Mdm36, forms a physical tether that robustly anchors mitochondria to the cell cortex but plays no direct role in mitochondrial division. Our analysis indicates that Num1-dependent anchoring is essential for distribution of the static mitochondrial network in fzo1 dnm1 cells. Consistently, expression of a synthetic mitochondria-cortex tether rescues the viability of fzo1 dnm1 num1 cells. We find that the cortical endoplasmic reticulum (ER) also is a constituent of the Num1 mitochondria-cortex tether, suggesting an active role for the ER in mitochondrial positioning in cells. Thus, taken together, our findings identify Num1 as a key component of a mitochondria-ER-cortex anchor, which we termed "MECA," that functions in parallel with mitochondrial dynamics to distribute and position the essential mitochondrial network.

  12. Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.

    PubMed

    Borradaile, Nica M; Han, Xianlin; Harp, Jeffrey D; Gale, Sarah E; Ory, Daniel S; Schaffer, Jean E

    2006-12-01

    Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 muM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing beta-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.

  13. ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants.

    PubMed

    Le Bars, Romain; Marion, Jessica; Le Borgne, Rémi; Satiat-Jeunemaitre, Béatrice; Bianchi, Michele Wolfe

    2014-06-20

    Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.

  14. (-)-Epicatechin mitigates high-fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress.

    PubMed

    Bettaieb, Ahmed; Vazquez Prieto, Marcela A; Rodriguez Lanzi, Cecilia; Miatello, Roberto M; Haj, Fawaz G; Fraga, César G; Oteiza, Patricia I

    2014-07-01

    We investigated the capacity of dietary (-)-epicatechin (EC) to mitigate insulin resistance through the modulation of redox-regulated mechanisms in a rat model of metabolic syndrome. Adolescent rats were fed a regular chow diet without or with high fructose (HFr; 10% w/v) in drinking water for 8 weeks, and a group of HFr-fed rats was supplemented with EC in the diet. HFr-fed rats developed insulin resistance, which was mitigated by EC supplementation. Accordingly, the activation of components of the insulin signaling cascade (insulin receptor, IRS1, Akt, and ERK1/2) was impaired, whereas negative regulators (PKC, IKK, JNK, and PTP1B) were upregulated in the liver and adipose tissue of HFr rats. These alterations were partially or totally prevented by EC supplementation. In addition, EC inhibited events that contribute to insulin resistance: HFr-associated increased expression and activity of NADPH oxidase, activation of redox-sensitive signals, expression of NF-κB-regulated proinflammatory cytokines and chemokines, and some sub-arms of endoplasmic reticulum stress signaling. Collectively, these findings indicate that EC supplementation can mitigate HFr-induced insulin resistance and are relevant for defining interventions that can prevent/mitigate MetS-associated insulin resistance.

  15. Lyn kinase represses mucus hypersecretion by regulating IL-13-induced endoplasmic reticulum stress in asthma.

    PubMed

    Wang, Xing; Yang, Xiaoqiong; Li, Yin; Wang, Xiaoyun; Zhang, Yun; Dai, Xi; Niu, Bin; Wu, Juan; Yuan, Xiefang; Xiong, Anjie; Liu, Zhigang; Zhong, Nanshan; Wu, Min; Li, Guoping

    2017-02-01

    In asthma, mucus hypersecretion is thought to be a prominent pathological feature associated with widespread mucus plugging. However, the current treatments for mucus hypersecretion are often ineffective or temporary. The potential therapeutic targets of mucus hypersecretion in asthma remain unknown. Here, we show that Lyn is a central effector of endoplasmic reticulum stress (ER stress) and mucous hypersecretion in asthma. In Lyn-transgenic mice (Lyn-TG) and wild-type (WT) C57BL/6J mice exposed to ovalbumin (OVA), Lyn overexpression attenuates mucus hypersecretion and ER stress. Interleukin 13 (IL-13) induced MUC5AC expression by enhancing ER stress in vitro. Lyn serves as a negative regulator of IL-13-induced ER stress and MUC5AC expression. We further find that an inhibitor of ER stress, which is likely involved in the PI3K p85α/Akt pathway and NFκB activity, blocked MUC5AC expression in Lyn-knockdown cells. Furthermore, PI3K/Akt signaling is required for IL-13-induced ER stress and MUC5AC expression in airway epithelial cells. The ER stress regulation of MUC5AC expression depends on NFκB in Lyn-knockdown airway epithelial cells. Our studies indicate not only a concept of mucus hypersecretion in asthma that involves Lyn kinase but also an important therapeutic candidate for asthma.

  16. Endoplasmic Reticulum Stress in Age-Related Macular Degeneration: Trigger for Neovascularization

    PubMed Central

    Salminen, Antero; Kauppinen, Anu; Hyttinen, Juha MT; Toropainen, Elisa; Kaarniranta, Kai

    2010-01-01

    Age-related macular degeneration (AMD) can be classified into two main categories: the atrophic, dry form and the exudative, wet form. The crucial difference between dry and wet AMD is the development of choroidal neovascularization in wet AMD. One fundamental cause of the neovascularization is the increased expression of VEGF (vascular endothelial growth factor) in retinal pigment epithelial cells. Progression of AMD is linked to augmentation of cellular stress, for example, oxidative stress, proteotoxic stress, inflammation and hypoxia. All these conditions can trigger stress in endoplasmic reticulum (ER), which maintains protein quality control in cells. ER stress induces the unfolded protein response (UPR) via IRE1 (inositol-requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) transducers. UPR signaling is a double-edged sword, that is, it can restore cellular homeostasis as far as possible, but ultimately may lead to chronic, overwhelming stress that can cause apoptotic cell death. Interestingly, ER stress is a well-known inducer of angiogenesis in cancer. Moreover, stress conditions associated with the progress of AMD can induce the expression of VEGF. We discuss the role of ER stress in the regulation of neovascularization and the conversion of dry AMD to its wet, detrimental counterpart. PMID:20683548

  17. Protective effect of mild endoplasmic reticulum stress on radiation-induced bystander effects in hepatocyte cells

    PubMed Central

    Xie, Yuexia; Ye, Shuang; Zhang, Jianghong; He, Mingyuan; Dong, Chen; Tu, Wenzhi; Liu, Peifeng; Shao, Chunlin

    2016-01-01

    Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the defense and self-protective mechanisms of bystander normal cells are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 cells under either normoxia or hypoxia, where the ratio of the yield of bystander MN induction to the yield of radiation-induced MN formation under hypoxia was much higher than that of normoxia. Nonetheless, thapsigargin induced endoplasmic reticulum (ER) stress and dramatically suppressed this bystander response manifested as the decrease of MN and apoptosis inductions. Meanwhile, the interference of BiP gene, a major ER chaperone, amplified the detrimental RIBE. More precisely, thapsigargin provoked ER sensor of PERK to initiate an instantaneous and moderate ER stress thus defensed the hazard form RIBE, while BiP depletion lead to persistently destroyed homeostasis of ER and exacerbated cell injury. These findings provide new insights that the mild ER stress through BiP-PERK-p-eIF2α signaling pathway has a profound role in protecting cellular damage from RIBE and hence may decrease the potential secondary cancer risk after cancer radiotherapy. PMID:27958308

  18. A neuroligin-4 missense mutation associated with autism impairs neuroligin-4 folding and endoplasmic reticulum export.

    PubMed

    Zhang, Chen; Milunsky, Jeff M; Newton, Stephanie; Ko, Jaewon; Zhao, Geping; Maher, Tom A; Tager-Flusberg, Helen; Bolliger, Marc F; Carter, Alice S; Boucard, Antony A; Powell, Craig M; Südhof, Thomas C

    2009-09-02

    Neuroligins (NLs) are postsynaptic cell-adhesion molecules essential for normal synapse function. Mutations in neuroligin-4 (NL4) (gene symbol: NLGN4) have been reported in some patients with autism spectrum disorder (ASD) and other neurodevelopmental impairments. However, the low frequency of NL4 mutations and the limited information about the affected patients and the functional consequences of their mutations cast doubt on the causal role of NL4 mutations in these disorders. Here, we describe two brothers with classical ASD who carry a single amino-acid substitution in NL4 (R87W). This substitution was absent from the brothers' asymptomatic parents, suggesting that it arose in the maternal germ line. R87 is conserved in all NL isoforms, and the R87W substitution is not observed in control individuals. At the protein level, the R87W substitution impaired glycosylation processing of NL4 expressed in HEK293 and COS cells, destabilized NL4, caused NL4 retention in the endoplasmic reticulum in non-neuronal cells and neurons, and blocked NL4 transport to the cell surface. As a result, the R87W substitution inactivated the synapse-formation activity of NL4 and abolished the functional effect of NL4 on synapse strength. Viewed together, these observations suggest that a point mutation in NL4 can cause ASD by a loss-of-function mechanism.

  19. Unique Matrix Structure in the Rough Endoplasmic Reticulum Cisternae of Pseudoachondroplasia Chondrocytes

    PubMed Central

    Merritt, Thomas M.; Bick, Roger; Poindexter, Brian J.; Alcorn, Joseph L.; Hecht, Jacqueline T.

    2007-01-01

    Mutations in cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Because COMP exists as a homopentamer, only one mutant COMP subunit may result in an abnormal complex that is accumulated in expanded rough endoplasmic reticulum (rER) cisternae, a hallmark of PSACH. Type IX collagen and matrilin-3 (MATN3), also accumulate in the rER cisternae of PSACH chondrocytes, but it is unknown how mutant COMP interacts with these proteins. The studies herein focus on defining the organization of these intracellularly retained proteins using fluorescence deconvolution microscopy. A unique matrix organization was identified in which type II procollagen formed a central core surrounded by a protein network of mutant COMP, type IX collagen, and MATN3. This pattern of matrix organization was found in multiple cisternae from single chondrocytes and in chondrocytes with different COMP mutations, indicating a common pattern of interaction. This suggests that stalling of mutant COMP and an interaction between mutant COMP and type II procollagen are initiating events in the assembly of matrix in the rER, possibly explaining why the material is not readily cleared from the rER. Altogether, these data suggest that mutant COMP initiates and perhaps catalyzes premature intracellular matrix assembly. PMID:17200202

  20. From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

    PubMed

    Farinha, Carlos M; Canato, Sara

    2017-01-01

    CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.